Inhibitors of the renal outer medullary potassium channel

ABSTRACT

This invention relates to compounds of Formula I having the following general structure: 
       Z 1 —Y 1 —(CH 2 ) n1 —R—(CH 2 ) n2 —Y 2 —Z 2  
 
     wherein R represents a fused bicyclic or spirocyclic aliphatic diamine and pharmaceutically acceptable salts thereof which are inhibitors of the Renal Outer Medullary Potassium (ROMK) channel (Kir1.1). The compounds are useful as diuretics and natriuretics and therefore are useful for the therapy and prophylaxis of disorders resulting from excessive salt and water retention, including cardiovascular diseases such as hypertension and chronic and acute heart failure.

BACKGROUND OF THE INVENTION

The Renal Outer Medullary Potassium (ROMK) channel (Kir1.1) (see e.g.,Ho, K., et al., Cloning and expression of an inwardly rectifyingATP-regulated potassium channel, Nature, 1993, 362(6415): p. 31-8.1, 2;and Shuck, M. E., et al., Cloning and characterization of multiple formsof the human kidney ROM-K potassium channel, J Biol Chem, 1994, 269(39):p. 24261-70) is a member of the inward rectifier family of potassiumchannels expressed in two regions of the kidney: thick ascending loop ofHenle (TALH) and cortical collecting duct (CCD) (see Hebert, S. C., etal., Molecular diversity and regulation of renal potassium channels,Physiol Rev, 2005, 85(1): p. 319-713). At the TALH, ROMK participates inpotassium recycling across the luminal membrane which is critical forthe function of the Na⁺/K⁺/2Cl⁻ co-transporter, the rate-determiningstep for salt reuptake in this part of the nephron. At the CCD, ROMKprovides a pathway for potassium secretion that is tightly coupled tosodium uptake through the amiloride-sensitive sodium channel (seeReinalter, S. C., et al., Pharmacotyping of hypokalaemic salt-losingtubular disorders, Acta Physiol Scand, 2004, 181(4): p. 513-21; andWang, W., Renal potassium channels: recent developments, Curr OpinNephrol Hypertens, 2004, 13(5): p. 549-55). Selective inhibitors of theROMK channel (also referred to herein as inhibitors of ROMK or ROMKinhibitors) are predicted to represent novel diuretics for the treatmentof hypertension and other conditions where treatment with a diureticwould be beneficial with potentially reduced liabilities (i.e., hypo- orhyperkalemia, new onset of diabetes, dyslipidemia) over the currentlyused clinical agents (see Lifton, R. P., A. G. Gharavi, and D. S.Geller, Molecular mechanisms of human hypertension, Cell, 2001, 104(4):p. 545-56). Human genetics (Ji, W., et al., Rare independent mutationsin renal salt handling genes contribute to blood pressure variation, NatGenet, 2008, 40(5): p. 592-9; and Tobin, M. D., et al., Common variantsin genes underlying monogenic hypertension and hypotension and bloodpressure in the general population, Hypertension, 2008, 51(6): p.1658-64) and genetic ablation of ROMK in rodents (see Lorenz, J. N., etal., Impaired renal NaCl absorption in mice lacking the ROMK potassiumchannel, a model for type II Bartter's syndrome, J Biol Chem, 2002,277(40): p. 37871-80 and Lu, M., et al., Absence of small conductance K+channel (SK) activity in apical membranes of thick ascending limb andcortical collecting duct in ROMK (Bartter's) knockout mice, J Biol Chem,2002, 277(40): p. 37881-7) support these expectations. To our knowledge,the first small molecule selective inhibitors of ROMK were reported fromwork done at Vanderbilt University as described in Lewis, L. M., et al.,High-Throughput Screening Reveals a Small-Molecule Inhibitor of theRenal Outer Medullary Potassium Channel and Kir7.1, Mol Pharmacol, 2009,76(5): p. 1094-1103. However, continuing discovery of selective smallmolecule inhibitors of ROMK is still needed for the development of newtreatments for hypertension and related disorders.

SUMMARY OF THE INVENTION

The present invention provides compounds of Formula I having the generalstructure below:

Z¹—Y¹—(CH₂)_(n1)—R—(CH₂)_(n2)—Y²—Z²  I

wherein R represents a fused bicyclic or spirocyclic aliphatic diamineand pharmaceutically acceptable salts thereof. The compounds of FormulaI are inhibitors of the ROMK (Kir1.1) channel and can thus act asdiuretics and natriuretics and are valuable pharmaceutically activecompounds for the therapy and prophylaxis of diseases, including, butnot limited to, cardiovascular diseases such as hypertension andconditions resulting from excessive salt and water retention. Methods oftreatment comprising administering a therapeutically or prophylacticallyeffective amount of a compound of Formula I to a patient in need of adiuretic and/or natriuretic agent are also provided. Compounds ofFormula I can be used in combination with other therapeuticallyeffective agents, including other drugs useful for the treatment ofhypertension and conditions resulting from excessive salt and waterretention. The invention furthermore relates to processes for preparingcompounds of Formula I, and pharmaceutical compositions which compriseany of the compounds of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of Formula I having the generalstructure below:

Z¹—Y¹—(CH₂)_(n1)—R—(CH₂)_(n2)—Y²—Z²  I

and pharmaceutically acceptable salts thereof wherein:n1 and n2 can be individually either 0 or 1;R represents a fused bicyclic or spirocyclic aliphatic diamine havingone of the following structures:

wherein R—I represents a 9-11-membered saturated bicyclic heterocyclicring system sharing 1 carbon atom, and R—II represents a 8-10-memberedsaturated bicyclic heterocyclic ring system sharing 2 carbon atoms,wherein each ring of R—I and each ring of R—II has 1 Nitrogen atom; R⁵is —H, —F (with the proviso that —F is not attached to a carbon that isalso attached to nitrogen), —CH₃, —CF₃, —CHF₂, —CH₂F, or —CH₂OH, or R⁵represents di-substitution on a single carbon with two of —F (with theproviso that —F is not attached to a carbon that is also attached tonitrogen) or two of —CH₃;

Z¹ is:

Z² is:

one of W¹ and W² is N and the other is CH;R¹ and R² are each independently —H, —F, —Cl, —Br, cyclopropyl,—C₁₋₃alkyl optionally substituted with 1-3 of —F, or —OC₁₋₃alkyloptionally substituted with 1-3 of —F;one of R^(3a) and R^(3b) is —CN, tetrazolyl, or —S(O)₂C₍₁₋₃₎alkyl andthe other is —H, —F, —Cl, —Br, —S—CH₃, —NH—CH₃, —O-cyclopropyl,—C₁₋₃alkyl optionally substituted with 1-3 of —F, or —OC₁₋₃alkyloptionally substituted with 1-3 of —F;one of R^(4a) and R^(4B) is CN, tetrazolyl, or —S(O)₂C₍₁₋₃₎alkyl and theother is —H, —F, —Cl, —Br, —S—CH₃, —NH—CH₃, —O-cyclopropyl, —C₁₋₃alkyloptionally substituted with 1-3 of —F, or —OC₁₋₃alkyl optionallysubstituted with 1-3 of —F;R^(a), R^(aa), R^(b) and R^(bb) are each independently —H, —F, —Cl,—C₁₋₃alkyl optionally substituted with 1 to 3 of F, or —OC₁₋₃alkyloptionally substituted with 1 to 3 of —F;R^(c) and R^(d) are each independently H, —F, —Cl, —C₁₋₃alkyl optionallysubstituted with 1 to 3 of F, or —OC₁₋₃alkyl optionally substituted with1 to 3 of —F; andone of Y¹ or Y² is —CH(OH)—; and the other is —CH(OH)—; —C(O)—; or—S(O)₂—; provided that where Y¹ or Y² is —C(O)— or —S(O)₂—, then theadjacent n1 or n2, respectively, is 0; and provided further that wheren1 or n2 is 0, the adjacent Y¹ or Y² is —C(O)— or —S(O)₂—.

In Embodiment A are compounds of Formula I and pharmaceuticallyacceptable salts thereof wherein R is R—I:

andwherein the other variables are as defined herein for Formula I.

In Embodiment B are compounds of Formula I and pharmaceuticallyacceptable salts thereof wherein R is R—II:

andwherein the other variables are as defined herein for Formula I.

In Embodiment C are compounds of Formula I and Embodiments A and B andpharmaceutically acceptable salts thereof wherein:

Z¹ is

and/or Z² is

wherein the other variables are as defined herein for Formula I. In aparticular subclass thereof, R¹ and/or R², as applicable, is —CH₃ andR^(c) and/or R^(d), as applicable, is —H. In another subclass thereof,Y¹ or Y² adjacent the foregoing isobenzofuran Z¹ and/or Z² is —CH(OH)—and the adjacent n1 or n2 is 1.

In Embodiment D are compounds of Formula I and Embodiments A-C andpharmaceutically acceptable salts thereof wherein:

Z¹ is

and/or

Z² is

wherein the other variables are as defined herein for Formula I. In aparticular subclass thereof, one of R^(3a) and R^(3b) is —CN,tetrazolyl, or —S(O)₂C₍₁₋₃₎alkyl and the other is —H, —F, —Cl, —Br,—C₁₋₃alkyl optionally substituted with 1-3 of —F, or —OC₁₋₃alkyloptionally substituted with 1-3 of —F and/or one of R^(4a) and R^(4b) is—CN, tetrazolyl, or —S(O)₂C₍₁₋₃₎alkyl and the other is —H, —F, —Cl, —Br,—C₁₋₃alkyl optionally substituted with 1-3 of —F, or —OC₁₋₃alkyloptionally substituted with 1-3 of —F. In another subclass thereof, Y¹or Y² adjacent the foregoing 6-membered Z¹ and/or Z² is —C(O)— or—S(O)₂— and the adjacent n1 or n2 is 0.

In Embodiment E are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

In Embodiment F are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

In Embodiment G are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

In Embodiment H are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

In Embodiment I are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

In Embodiment J are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

In Embodiment K are compounds of Formula I and Embodiments A-D andpharmaceutically acceptable salts thereof wherein R is:

wherein the other variables are as defined herein for Formula I.

The following select embodiments of Formula I are exemplified herein.These compounds and their pharmaceutically acceptable salts formindividual embodiments of the present invention:

-   5,5′-{2,7-Diazaspiro[4.5]decane-2,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)    [EXAMPLE 1];-   5-((1R)-2-(7-(6-(1H-tetrazol-1-yl)nicotinoyl)-2,7-diazaspiro[4.5]decan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 14];-   5,5′-{2,8-diazaspiro[4.5]decane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)    [EXAMPLE 2];-   6-({2-[(2R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]octahydro-5H-pyrrolo[3,4-c]pyridine-5-yl}carbonyl)pyridine-3-carbonitrile    [EXAMPLE 3];-   5-((1R)-2-(5-(6-(1H-Tetrazol-1-yl)nicotinoyl)-1H-pyrrolo[3,4-c]_yridine-2(3H,3aH,4H,5H,6H,7H,7aH)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 15];-   5,5′-{1,7-Diazaspiro[4.5]decane-1,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)    [EXAMPLE 5];-   5,5′-{2,8-Diazaspiro[5.5]undecane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)    [EXAMPLE 6];-   5-((1R)-2-(8-(6-(1H-Tetrazol-1-yl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 13];-   5-((1R)-1-Hydroxy-2-(8-(6-(methylsulfonyl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)ethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 21];-   5,5′-{Hexahydropyrrolo[3,4-b]pyrrole-1,5-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one    [EXAMPLE 4];-   6-(1-Hydroxy-2-{5-[(2R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl}ethyl)pyridine-3-carbonitrile    [EXAMPLE 7];-   4-(1-((R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-5(1H)-ylsulfonyl)benzonitrile    [EXAMPLE 8];-   4-(5-((R)-2-Hydroxy-2-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-ylsulfonyl)benzonitrile    [EXAMPLE 9];-   6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrile    [EXAMPLE 11];-   6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrile    [EXAMPLE 12];-   5-((1R)-2-(5-(5-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 16];-   5-((1R)-2-(5-(6-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 17];-   5-((1R)-2-(5-(4-(1H-Tetrazol-1-yl)phenylsulfonyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 20];-   5,5′-(1R,1′R)-2,2′-(1,7-Diazaspiro[4.4]nonane-1,7-diyl)bis(1-hydroxyethane-2,1-diyl)bis(4-methylisobenzofuran-1(3H)-one)    [EXAMPLE 10];-   5-((1R)-1-Hydroxy-2-(1-(3-methyl-4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 18];-   5-((1R)-1-Hydroxy-2-(1-(4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-one    [EXAMPLE 19];-   6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrile    [EXAMPLE 22];-   6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrile    [EXAMPLE 23];-   6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrile    [EXAMPLE 24]; and-   6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrile    [EXAMPLE 25].

All structural Formulas and embodiments described herein include thepharmaceutically acceptable salts thereof.

As used herein except if noted otherwise, “alkyl” is intended to includeboth branched- and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms. Commonly used abbreviationsfor alkyl groups are used throughout the specification. For example theterm “C₁₋₆ alkyl” (or “C₁-C₆ alkyl”), means linear or branched chainalkyl groups, including all isomers, having the specified number ofcarbon atoms and includes all of the hexyl and pentyl isomers as well asn-, iso-, sec- and tert-butyl (butyl, s-butyl, i-butyl, t-butyl;Bu=butyl), n- and i-propyl (Pr=propyl), ethyl (Et) and methyl (Me).

“Cycloalkyl” is a cyclized alkyl ring having the indicated number ofcarbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl. The cycloalkyl ring may be substituted onany available carbon which results in the creation of a stablestructure, including the ring carbon which serves as the point ofattachment to the rest of the molecule.

Halo or halogen refers to —F (fluoro), —Cl (chloro), —Br (bromo) and —I(iodo). Preferred halogens are —F and —Cl.

Unless expressly depicted or described otherwise, variables depicted ina structural formula with a “floating” bond, such as each ofsubstituents R⁵, R^(a), R^(aa), R^(b), R^(bb), R^(c) and R^(d) instructural Formula I, are permitted on any available carbon atom in thering to which each is attached provided they are not on a carbon at thespiro junction within R—I. For purposes of exemplification and withoutlimitation, if the structure was as follows:

substitution can be on any carbon labeled as positions 1-6, with theproviso that the substitution can not be on the carbon at the spirojunction within R—I. In R—II structures, substitution can be on theshared carbons.

Optional substitution on a chemical moiety encompasses the presence orabsence of substituents on the specified moiety. For example, —C₁₋₃alkyl optionally substituted with 1-3 of —F describes unsubstituted—C₁₋₃ alkyl (e.g., —CH₃, —CH₂CH₃, —(CH₂)₂CH₃, or —CH(CH₃)₂, orfluoro-substituted —C₁₋₃ alkyl including but not limited to —CH₂F,—CHF₂, —CF₃, or —CH₂CF₃.

The present invention encompasses all stereoisomeric forms of thecompounds of Formula I. Centers of asymmetry that are present in thecompounds of Formula I can all independently of one another have (R)configuration or (S) configuration. When bonds to the chiral carbon aredepicted as straight lines in the structural Formulas of the invention,it is understood that both the (R) and (S) configurations of the chiralcarbon, or when a compound name is recited without a chiral designationfor a chiral carbon, it is understood that both the (R) and (S)configurations of the chiral carbon, and hence both enantiomers andmixtures thereof, are embraced within the Formula or by the name. Theproduction of specific stereoisomers or mixtures thereof may beidentified in the Examples where such stereoisomers or mixtures wereobtained, but this in no way limits the inclusion of all stereoisomersand mixtures thereof from being within the scope of this invention.

The invention includes all possible enantiomers and diastereomers andmixtures of two or more stereoisomers, for example mixtures ofenantiomers and/or diastereomers, in all ratios. Thus, enantiomers are asubject of the invention in enantiomerically pure form, both aslevorotatory and as dextrorotatory antipodes, in the form of racematesand in the form of mixtures of the two enantiomers in all ratios. In thecase of a cis/trans isomerism the invention includes both the cis formand the trans form as well as mixtures of these forms in all ratios. Thepreparation of individual stereoisomers can be carried out, if desired,by separation of a mixture by customary methods, for example bychromatography or crystallization, by the use of stereochemicallyuniform starting materials for the synthesis or by stereoselectivesynthesis. Optionally a derivatization can be carried out before aseparation of stereoisomers. The separation of a mixture ofstereoisomers can be carried out at an intermediate step during thesynthesis of any compound of Formula I or it can be done on a finalracemic product. Absolute stereochemistry may be determined by X-raycrystallography of crystalline products or crystalline intermediateswhich are derivatized, if necessary, with a reagent containing astereogenic center of known configuration. Where compounds of thisinvention are capable of tautomerization, all individual tautomers aswell as mixtures thereof are included in the scope of this invention.The present invention includes all such isomers, as well as salts,solvates (including hydrates) and solvated salts of such racemates,enantiomers, diastereomers and tautomers and mixtures thereof.

Reference to the compounds of Formula I herein encompasses the compoundsof Formula I and all embodiments thereof. Reference to the compounds ofthis invention as those of a specific formula or embodiment, e.g.,Formula I and embodiments thereof, or any other generic structuralformula or specific compound described or claimed herein, is intended toencompass the specific compound or compounds falling within the scope ofthe formula or embodiment, including salts thereof, particularlypharmaceutically acceptable salts, solvates of such compounds andsolvated salt forms thereof, where such forms are possible unlessspecified otherwise.

In the compounds of Formula I, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of Formula I. Forexample, different isotopic forms of hydrogen (H) include protium (¹H)and deuterium (²H). Protium is the predominant hydrogen isotope found innature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples. Isotopically-enriched compoundswithin Formula I can be prepared without undue experimentation byconventional techniques well known to those skilled in the art or byprocesses analogous to those described in the Schemes and Examplesherein using appropriate isotopically-enriched reagents and/orintermediates.

When the compounds of Formula I contain one or more acidic or basicgroups the invention also includes the corresponding pharmaceuticallyacceptable salts. Thus, the compounds of Formula I which contain acidicgroups can be used according to the invention, for example, as alkalimetal salts, alkaline earth metal salts or as ammonium salts. Examplesof such salts include but are not limited to sodium salts, potassiumsalts, calcium salts, magnesium salts or salts with ammonia or organicamines such as, for example, ethylamine, ethanolamine, triethanolamineor amino acids. Compounds of Formula I which contain one or more basicgroups, i.e. groups which can be protonated, can be used according tothe invention in the form of their acid addition salts with inorganic ororganic acids as, for example but not limited to, salts with hydrogenchloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid,benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acids, oxalic acid, acetic acid, trifluoroaceticacid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formicacid, propionic acid, pivalic acid, diethylacetic acid, malonic acid,succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid,isonicotinic acid, citric acid, adipic acid, etc. If the compounds ofFormula I simultaneously contain acidic and basic groups in the moleculethe invention also includes, in addition to the salt forms mentioned,inner salts or betaines (zwitterions). Salts can be obtained from thecompounds of Formula I by customary methods which are known to theperson skilled in the art, for example by combination with an organic orinorganic acid or base in a solvent or dispersant, or by anion exchangeor cation exchange from other salts. The present invention also includesall salts of the compounds of Formula I which, owing to lowphysiological compatibility, are not directly suitable for use inpharmaceuticals but which can be used, for example, as intermediates forchemical reactions or for the preparation of physiologically (i.e.,pharmaceutically) acceptable salts.

Furthermore, compounds of the present invention may exist in amorphousform and/or one or more crystalline forms, and as such all amorphous andcrystalline forms and mixtures thereof of the compounds of Formula I areintended to be included within the scope of the present invention. Inaddition, some of the compounds of the instant invention may formsolvates with water (i.e., a hydrate) or common organic solvents. Suchsolvates and hydrates, particularly the pharmaceutically acceptablesolvates and hydrates, of the instant compounds are likewise encompassedwithin the scope of this invention, along with un-solvated and anhydrousforms.

Any pharmaceutically acceptable pro-drug modification of a compound ofthis invention which results in conversion in vivo to a compound withinthe scope of this invention is also within the scope of this invention.For example, esters can optionally be made by esterification of anavailable carboxylic acid group or by formation of an ester on anavailable hydroxy group in a compound. Similarly, labile amides can bemade. Pharmaceutically acceptable esters or amides of the compounds ofthis invention may be prepared to act as pro-drugs which can behydrolyzed back to an acid (or —COO— depending on the pH of the fluid ortissue where conversion takes place) or hydroxy form particularly invivo and as such are encompassed within the scope of this invention.Examples of pharmaceutically acceptable pro-drug modifications include,but are not limited to, —C₁₋₆alkyl esters and —C₁₋₆alkyl substitutedwith phenyl esters.

Accordingly, the compounds within the generic structural formulas,embodiments and specific compounds described and claimed hereinencompass salts, all possible stereoisomers and tautomers, physicalforms (e.g., amorphous and crystalline forms), solvate and hydrate formsthereof and any combination of these forms, as well as the saltsthereof, pro-drug forms thereof, and salts of pro-drug forms thereof,where such forms are possible unless specified otherwise.

The compounds of Formula I according to the invention are inhibitors ofROMK, and are therefore useful as diuretic and/or natriuretic agents.ROMK inhibitors help to increase urination and increase urine volume andalso to prevent or reduce reabsorption of sodium in the kidneys leadingto increased excretion of sodium and water. Therefore, the compounds areuseful for treatment or prophylaxis of disorders that benefit fromincreased excretion of water and sodium from the body. Accordingly, thepresent invention provides a method for inhibiting ROMK comprisingadministering a compound of Formula I in a ROMK-inhibitory effectiveamount to a patient in need thereof. The inhibition of ROMK by thecompounds of Formula I can be examined, for example, in any of theactivity assays described below. The invention also provides a methodfor causing diuresis, natriuresis or both, comprising administering acompound of Formula I in a therapeutically effective amount to a patientin need thereof.

Due to their activity as diuretics and natriuretic agents, thisinvention further provides the use of compounds of Formula I in methodsfor treatment of, prevention of or reduction of risk for developingmedical conditions that benefit from increased excretion of water andsodium, such as but not limited to one or more of hypertension, heartfailure (both acute and chronic, the latter also known as congestiveheart failure) and/or other conditions resulting from excessive salt andwater retention. It further includes the use of the compounds of FormulaI in methods for treatment of, prevention of or reduction of risk fordeveloping one or more disorders such as pulmonary arterial hypertension(PAH), cardiovascular disease, diabetes mellitus, diabetes insipidus,post-operative volume overload, endothelial dysfunction, diastolicdysfunction, systolic dysfunction, stable and unstable angina pectoris,thromboses, restenosis, myocardial infarction, stroke, cardiacinsufficiency, pulmonary hypertonia, atherosclerosis, hepatic cirrhosis,ascitis, pre-eclampsia, cerebral edema, nephropathy, glomerulonephritis,nephrotic syndrome, acute and chronic kidney insufficiency, acutetubular necrosis, hypercalcemia, idiopathic edema, Dent's disease,Meniere's disease, edetamous states, glaucoma, benign intracranialhypertension, and other conditions for which a diuretic would havetherapeutic or prophylactic benefit. The compounds of the invention canbe administered to a patient having, or at risk of having, one or moreconditions for which a diuretic would have therapeutic or prophylacticbenefit such as those described herein.

In general, compounds that are ROMK inhibitors can be identified asthose compounds which, when tested, have an IC₅₀ of 5 μM or less,preferably 1 μM or less, and more preferably 0.25 μM or less, in atleast one of the following assays: 1) the Electrophysiology Assay and 2)the Thallium Flux Assay. These assays are described in more detailfurther below.

The dosage amount of the compound to be administered depends on theindividual case and is, as is customary, to be adapted to the individualcircumstances to achieve an optimum effect. Thus, it depends on thenature and the severity of the disorder to be treated, and also on thesex, age, weight and individual responsiveness of the human or animal tobe treated, on the efficacy and duration of action of the compoundsused, on whether the therapy is acute or chronic or prophylactic, or onwhether other active compounds are administered in addition to thecompounds of Formula I. A consideration of these factors is well withinthe purview of the ordinarily skilled clinician for the purpose ofdetermining the therapeutically effective or prophylactically effectivedosage amount needed to prevent, counter, or arrest the progress of thecondition. It is expected that the compound will be administeredchronically on a daily basis for a length of time appropriate to treator prevent the medical condition relevant to the patient, including acourse of therapy lasting days, months, years or the life of thepatient.

In general, a daily dose of approximately 0.001 to 100 mg/kg, preferably0.001 to 30 mg/kg, in particular 0.001 to 10 mg/kg (in each case mg perkg of bodyweight) is appropriate for administration to an adult weighingapproximately 75 kg in order to obtain the desired results. The dailydose is preferably administered in a single dose or can be divided intoseveral, for example two, three or four individual doses, and may be,for example but not limited to, 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg,1.25 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 20 mg, 40 mg, 50 mg, 75 mg, 100 mg,125 mg, 150 mg, 175 mg, 200 mg, etc., on a daily basis. In some cases,depending on the individual response, it may be necessary to deviateupwards or downwards from the given daily dose. Furthermore, thecompound may be formulated for immediate or modified release such asextended or controlled release.

The term “patient” includes animals, preferably mammals and especiallyhumans, who use the instant active agents for the prophylaxis ortreatment of a medical condition. Administering of the drug to thepatient includes both self-administration and administration to thepatient by another person. The patient may be in need of treatment foran existing disease or medical condition, or may desire prophylactictreatment to prevent or reduce the risk for developing said disease ormedical condition or developing long-term complications from a diseaseor medical condition.

The term therapeutically effective amount is intended to mean thatamount of a drug or pharmaceutical agent that will elicit the biologicalor medical response of a tissue, a system, animal or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician.A prophylactically effective amount is intended to mean that amount of apharmaceutical drug that will prevent or reduce the risk of occurrenceof the biological or medical event that is sought to be prevented in atissue, a system, animal or human by a researcher, veterinarian, medicaldoctor or other clinician. It is understood that a specific daily dosageamount can simultaneously be both a therapeutically effective amount,e.g., for treatment of hypertension, and a prophylactically effectiveamount, e.g., for prevention or reduction of risk of myocardialinfarction or prevention and reduction of risk for complications relatedto hypertension.

In the methods of treatment of this invention, the ROMK inhibitors maybe administered via any suitable route of administration such as, forexample, orally, parenterally, or rectally in dosage unit formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants and vehicles. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Oral formulations are preferred fortreatment of chronic indications such as hypertension or chronic heartfailure, particularly solid oral dosage units such as pills, tablets orcapsules, and more particularly tablets. IV dosing is preferred foracute treatment, for example, for the treatment of acute heart failure.

This invention also provides pharmaceutical compositions comprised of acompound of Formula I and a pharmaceutically acceptable carrier which iscomprised of one or more excipients or additives. An excipient oradditive is an inert substance used to formulate the active drugingredient. For oral use, the pharmaceutical compositions of thisinvention containing the active ingredient may be in forms such aspills, tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or soft capsules, orsyrups or elixirs. Compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients, whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, mannitol, calcium phosphate or sodium phosphate; granulatingand disintegrating agents, for example, corn starch, or alginic acid;binding agents, for example starch, gelatin or acacia, and lubricatingagents, for example, magnesium stearate, stearic acid or talc.

Pharmaceutical compositions may also contain other customary additives,for example, wetting agents, stabilizers, emulsifiers, dispersants,preservatives, sweeteners, colorants, flavorings, aromatizers,thickeners, buffer substances, solvents, solubilizers, agents forachieving a depot effect, salts for altering the osmotic pressure,coating agents or antioxidants.

Oral immediate-release and time-controlled release dosage forms may beemployed, as well as enterically coated oral dosage forms. Tablets maybe uncoated or they may be coated by known techniques for aestheticpurposes, to mask taste or for other reasons. Coatings can also be usedto delay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredients is mixed with water ormiscible solvents such as propylene glycol, PEGs and ethanol, or an oilmedium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Oilysuspensions may be formulated by suspending the active ingredient in avegetable oil, for example arachis oil, olive oil, sesame oil or coconutoil, or in mineral oil such as liquid paraffin. The oily suspensions maycontain a thickening agent, for example beeswax, hard paraffin or cetylalcohol. Sweetening agents and flavoring agents may be added to providea palatable oral preparation. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid. Syrups and elixirsmay be formulated with sweetening agents, for example glycerol,propylene glycol, sorbitol or sucrose.

The instant invention also encompasses a process for preparing apharmaceutical composition comprising combining a compound of Formula Iwith a pharmaceutically acceptable carrier. Also encompassed is thepharmaceutical composition which is made by combining a compound ofFormula I with a pharmaceutically acceptable carrier. Furthermore, atherapeutically effective amount of a compound of this invention can beused for the preparation of a medicament useful for inhibiting ROMK, forcausing diuresis and/or natriuresis, and/or for treating, preventing orreducing the risk for any of the medical conditions described herein, indosage amounts described herein.

The amount of active compound of Formula I and/or its pharmaceuticallyacceptable salts in the pharmaceutical composition may be, for examplebut not limited to, from 0.1 to 200 mg, particularly from 0.1 to 100 mg,and more particularly from 0.1 to 50 mg, per dose on a free acid/freebase weight basis, but depending on the type of the pharmaceuticalcomposition, potency of the active ingredient and/or the medicalcondition being treated, it could also be lower or higher.Pharmaceutical compositions usually comprise 0.5 to 90 percent by weightof the active compound on a free acid/free base weight basis.

The compounds of Formula I inhibit ROMK. On account of this property,apart from use as pharmaceutically active compounds in human medicineand veterinary medicine, they can also be employed as a scientific toolor as aid for biochemical investigations in which such an effect on ROMKis intended, and also for diagnostic purposes, for example in the invitro diagnosis of cell samples or tissue samples. The compounds ofFormula I can also be employed as intermediates for the preparation ofother pharmaceutically active compounds.

One or more additional pharmacologically active agents may beadministered in combination with a compound of Formula I. An additionalactive agent (or agents) is intended to mean a compound that isdifferent from the compound of Formula I, and which is apharmaceutically active agent (or agents) that is active in the body,including pro-drugs that convert to pharmaceutically active form afteradministration, and also includes free-acid, free-base, andpharmaceutically acceptable salts of said additional active agents whensuch forms are sold commercially or are otherwise chemically possible.Generally, any suitable additional active agent or agents, including butnot limited to anti-hypertensive agents, additional diuretics,anti-atherosclerotic agents such as a lipid modifying compound,anti-diabetic agents and/or anti-obesity agents may be used in anycombination with the compound of Formula I in a single dosageformulation (a fixed dose drug combination), or may be administered tothe patient in one or more separate dosage formulations which allows forconcurrent or sequential administration of the active agents(co-administration of the separate active agents). Examples ofadditional active agents which may be employed include but are notlimited to angiotensin converting enzyme inhibitors (e.g, alacepril,benazepril, captopril, ceronapril, cilazapril, delapril, enalapril,enalaprilat, fosinopril, imidapril, lisinopril, moveltipril,perindopril, quinapril, ramipril, spirapril, temocapril, ortrandolapril), angiotensin II receptor antagonists also known asangiotensin receptor blockers or ARBs (e.g., losartan i.e., COZAAR®,valsartan, candesartan, olmesartan, telmesartan, eprosartan, irbesartan,azilsartan and any of these drugs used in combination with thiazide-likediuretics such as hydrochlorothiazide such as HYZAAR®), diuretics, e.g.,hydrochlorothiazide (HCTZ); potassium sparing diuretics such asamiloride HCl, spironolactone, epleranone, triamterene, each with orwithout HCTZ; carbonic anhydrase inhibitors, such as acetazolamide,neutral endopeptidase inhibitors (e.g., thiorphan and phosphoramidon),aldosterone antagonists, aldosterone synthase inhibitors, renininhibitors (e.g. urea derivatives of di- and tri-peptides (See U.S. Pat.No. 5,116,835), amino acids and derivatives (U.S. Pat. Nos. 5,095,119and 5,104,869), amino acid chains linked by non-peptidic bonds (U.S.Pat. No. 5,114,937), di- and tri-peptide derivatives (U.S. Pat. No.5,106,835), peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079) and peptidyl beta-aminoacyl aminodiol carbamates (U.S. Pat.No. 5,089,471); also, a variety of other peptide analogs as disclosed inthe following U.S. Pat. Nos. 5,071,837; 5,064,965; 5,063,207; 5,036,054;5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors(including diol sulfonamides and sulfinyls (U.S. Pat. No. 5,098,924),N-morpholino derivatives (U.S. Pat. No. 5,055,466), N-heterocyclicalcohols (U.S. Pat. No. 4,885,292) and pyrolimidazolones (U.S. Pat. No.5,075,451); also, pepstatin derivatives (U.S. Pat. No. 4,980,283) andfluoro- and chloro-derivatives of statone-containing peptides (U.S. Pat.No. 5,066,643), enalkrein, RO 42-5892, A 65317, CP 80794, ES 1005, ES8891, SQ 34017, aliskiren(2(S),4(S),5(S),7(S)—N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamidhemifumarate) SPP600, SPP630 and SPP635), endothelin receptorantagonists, mineralocorticoid receptor antagonists, vasodilators (e.g.nitroprusside), calcium channel blockers (e.g., amlodipine, nifedipine,veraparmil, diltiazem, felodipine, gallopamil, niludipine, nimodipine,nicardipine), potassium channel activators (e.g., nicorandil, pinacidil,cromakalim, minoxidil, aprilkalim, loprazolam), sympatholitics,beta-adrenergic blocking drugs (e.g., acebutolol, atenolol, betaxolol,bisoprolol, carvedilol, metoprolol, metoprolol tartate, nadolol,propranolol, sotalol, timolol), alpha adrenergic blocking drugs (e.g.,doxazocin, prazocin or alpha methyldopa); central alpha adrenergicagonists; peripheral vasodilators (e.g. hydralazine); nitrates or nitricoxide donating compounds, e.g., isosorbide mononitrate, lipid loweringagents (e.g., HMG-CoA reductase inhibitors such as simvastatin andlovastatin which are marketed as ZOCOR® and MEVACOR® in lactone pro-drugform and function as inhibitors after administration, andpharmaceutically acceptable salts of dihydroxy open ring acid HMG-CoAreductase inhibitors such as atorvastatin (particularly the calcium saltsold in LIPITOR®, rosuvastatin (particularly the calcium salt sold inCRESTOR®), pravastatin (particularly the sodium salt sold inPRAVACHOL®), and fluvastatin (particularly the sodium salt sold inLESCOL®); a cholesterol absorption inhibitor such as ezetimibe (ZETIA®),and ezetimibe in combination with any other lipid lowering agents suchas the HMG-CoA reductase inhibitors noted above and particularly withsimvastatin (VYTORIN®) or with atorvastatin calcium; niacin inimmediate-release or controlled release forms, and particularly niacinin combination with a DP antagonist such as laropiprant (TREDAPTIVE®)and/or with an HMG-CoA reductase inhibitor; niacin receptor agonistssuch as acipimox and acifran, as well as niacin receptor partialagonists; metabolic altering agents including insulin sensitizing agentsand related compounds for the treatment of diabetes such as biguanides(e.g., metformin), meglitinides (e.g., repaglinide, nateglinide),sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide,tolazamide, tolbutamide), thiazolidinediones also referred to asglitazones (e.g., pioglitazone, rosiglitazone), alpha glucosidaseinhibitors (e.g., acarbose, miglitol), dipeptidyl peptidase inhibitors,e.g., (sitagliptin (JANUVIA®), alogliptin, vildagliptin, saxagliptin,linagliptin, dutogliptin, gemigliptin), ergot alkaloids (e.g.,bromocriptine), combination medications such as JANUMET® (sitagliptinwith metformin), and injectable diabetes medications such as exenatideand pramlintide acetate; or with other drugs beneficial for theprevention or the treatment of the above-mentioned diseases includingbut not limited to diazoxide; and including the free-acid, free-base,and pharmaceutically acceptable salt forms of the above active agentswhere chemically possible.

Several methods for preparing the compounds of this invention aredescribed in the following Schemes and Examples. Starting materials andintermediates are purchased from commercial sources, made from knownprocedures, or are otherwise illustrated. In some cases the order ofcarrying out the steps of the reaction schemes may be varied tofacilitate the reaction or to avoid unwanted reaction products. The Argroup shown in the below schemes can represent any of themono-or-bi-cyclic rings at the terminal end of Z¹ or Z² as definedpreviously.

Synthesis of the compounds disclosed herein is generally provided for inthe following schemes.

The preparation of the compounds I1 is detailed in Scheme 1. Treatmentof the styrene epoxide 1-1 with an appropriate monoprotected diamine 1-2under appropriate coupling conditions (such as heating in alcoholicsolvent, or microwave heating) affords the amino alcohol product 1-3.The Boc protecting group (Greene, T.; Wuts, P. G. M. Protective Groupsin Organic Synthesis, John Wiley and Sons, Inc., New York, N.Y. 1991) of1-3 can be removed under acidic conditions, such as with TFA or HCl.Alternatively, the diamine may be protected with another protectinggroup such as Cbz, and subsequently removed by hydrogenolysis. Foroptimal regioselectivity in the epoxide opening the free base of theresulting amine should be generated in situ (as described in thepreparation of EXAMPLE 1, for instance) or isolated previously throughstandard methods (for example sodium carbonate wash and extraction, ionexchange column chromatography, etc.). The resulting amine may becoupled to another epoxide 1-5 (which may or may not be the same as 1-1)under the conditions described above to provide compounds I1.

Additionally, compounds of formula I2 can also be prepared by thesequence detailed in Scheme 2. Treatment of the previously describedintermediate 1-4 with the appropriate electrophile 2-1 (such ascarboxylic acid or ester) under standard amide bond forming conditions(such as EDC, HOBt, triethylamine) gives rise to I2.

The preparation of compounds of formula I3 is shown in Scheme 3. Again,starting from intermediate 1-4, coupling with the appropriate sulfonicacid or activated derivative (such as sulfonyl chloride) underappropriate conditions (such as triethylamine) provides the sulfonamidesI3.

General Procedures.

The independent synthesis of diastereomers and enantiomers or theirchromatographic separations may be achieved as known in the art byappropriate modification of the methodology disclosed herein. Theirabsolute stereochemistry may be determined by x-ray crystallography ofcrystalline products or crystalline intermediates which are derivatized,if necessary, with a reagent containing an asymmetric center of knownabsolute stereochemistry.

The subject compounds may be prepared by modification of the proceduresdisclosed in the Examples as appropriate. Starting materials arecommercially available or made by known procedures or as illustrated.The following examples are provided for the purpose of furtherillustration only and are not intended to be limitations of thedisclosed invention.

Reactions sensitive to moisture or air were performed under nitrogen orargon using anhydrous solvents and reagents. The progress of reactionswas determined by either analytical thin layer chromatography (TLC)usually performed with E. Merck precoated TLC plates, silica gel60E-254, layer thickness 0.25 mm or liquid chromatography-massspectrometry (LC-MS; also referred to as “LC” in the experimentalprocedures herein).

Typically the analytical LC-MS system used consisted of a Waters® ZQ™platform with electrospray ionization in positive ion detection modewith an Agilent® 1100 series HPLC with autosampler. The column wasusually a Waters® Xterra® MS C18, 3.0×50 mm, 5 μm. The flow rate was 1mL/minute, and the injection volume was 10 μL. UV detection was in therange of 210-400 nm. The mobile phase consisted of solvent A (water plus0.06% TFA) and solvent B (acetonitrile plus 0.05% TFA) with a gradientof 100% solvent A for 0.7 minutes changing to 100% solvent B over 3.75minutes, maintained for 1.1 minutes, then reverting to 100% solvent Aover 0.2 minutes.

Preparative High Performance Liquid Chromatography (HPLC) purificationswere usually performed using a mass spectrometry directed system.Usually they were performed on a Waters® Chromatography Workstationconfigured with LC-MS System Consisting of: Waters® ZQ™ single quad MSsystem with Electrospray Ionization, Waters® 2525 Gradient Pump, Waters®2767 Injector/Collector, Waters® 996 PDA Detector, the MS Conditions of:150-750 amu, Positive Electrospray, Collection Triggered by MS, and aWaters® Sunfire C-18 5 micron, 30 mm (id)×100 mm column. The mobilephases consisted of mixtures of acetonitrile (10-100%) in watercontaining 0.1% TFA. Flow rates were maintained at 50 mL/minute, theinjection volume was 1800 μL, and the UV detection range was 210-400 nm.Mobile phase gradients were optimized for the individual compounds.

Reactions performed using microwave irradiation were normally carriedout using an Emrys™ Optimizer manufactured by Personal Chemistry, or anInitiator manufactured by Biotage.

Concentration of solutions was carried out on a rotary evaporator underreduced pressure. Flash chromatography was usually performed using aBiotage Flash Chromatography apparatus (Dyax Corp.) on silica gel (32-63mM, 60 Å pore size) in pre-packed cartridges of the size noted. ¹H NMRspectra were acquired at 500 MHz spectrometers in CDCl₃ solutions unlessotherwise noted. Chemical shifts were reported in parts per million(ppm). Tetramethylsilane (TMS) was used as internal reference in CD₃C1solutions, and residual CH₃OH peak or TMS was used as internal referencein CD₃OD solutions. Coupling constants (J) were reported in hertz (Hz).Chiral analytical chromatography was performed on one of Chiralpak® AS,Chiralpak® AD, Chiralcel® OD, Chiralcel® IA, or Chiralcel® OJ columns(250×4.6 mm) (Daicel Chemical Industries, Ltd.) with noted percentage ofeither ethanol in hexane (% Et/Hex) or isopropanol in heptane (%IPA/Hep) as isocratic solvent systems. Chiral preparative chromatographywas conducted on one of Chiralpak® AS, Chiralpak® AD, Chiralcel® OD,Ciralcel® IA, or Chiralcel® OJ columns (20×250 mm) (Daicel ChemicalIndustries, Ltd.) with desired isocratic solvent systems identified onchiral analytical chromatography or by supercritical fluid (SFC)conditions.

In the Examples, when a compound is obtained via chromatography (e.g.,MPLC, HPLC, silica gel), it means that the solvent was removed(generally under vacuum) after the chromatography step to obtain theisolated product.

Abbreviations usd herein include: —C(O)CH₃ (Ac); acetic acid (AcOH);—OC(O)CH₃ (OAc); aqueous (aq); Cbz (benzyloxycarbonyl);N;N-diisopropylethylamine (DIEA); N;N-dimethylformamide (DMF); ethylacetate (EtOAc); diethyl ether (ether or Et₂O); petroleum ether (PetEther; PE); gram(s) (g); hour(s) (h or hr); 2-propanol (IPA); massspectrum (ms or MS); microliter(s) (4); milligram(s) (mg); milliliter(s)(mL); millimole (mmol); minute(s) (min); methyl t-butylether (MTBE);(benzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate(PyBOP); retention time (R_(t)); room temperature (rt or RT); saturatedaq sodium chloride solution (brine); trifluoroacetic acid (TFA);triethylamine (TEA); hydrochloric acid (HCl); tetrahydrofuran (THF);flash chromatography (FC); medium pressure liquid chromatography (MPLC);liquid chromatography (LC); liquid chromatography-mass spectrometry(LCMS or LC-MS); supercritical fluid chromatography (SFC);t-butyloxycarbonyl (Boc or BOC); Diethylaminosulfur trifluoride (DAST);dichloromethane (DCM); dimethylacetamide (DMA; DMAC); dimethylsulfoxide(DMSO); 1,3-Bis(diphenylphosphino)propane (DPPP); acetic acid (HOAc);3-chloroperoxybenzoic acid (m-CPBA); methyl (Me); methanol (MeOH);N-bromosuccinamide (NBS); thin layer chromatography (TLC);N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC); roundbottom (RB); diisopropylamine (DIPA); hexamethylphosphoramide (HMPA);1-hydroxybenzotriazole (HOBt); lithium diisopropylamide (LDA); highperformance liquid chromatography/mass spectrometry (HPLC-MS);nicotinamide adenine dinucleotide (NADP); isopropyl acetate (IPAc);0-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU). Celite® is a tradename for diatomaceous earth.

The following are representative procedures for the preparation of thecompounds used in the following Examples, or which can be substitutedfor the compounds used in the following Examples which may not becommercially available.

Intermediate 1

5-Bromo-4-Methyl-2-benzofuran-1(3H)-one Step A:(3-Bromo-2-methylphenyl)methanol

To a solution of 3-bromo-2-methyl benzoic acid (35 g, 160 mmol) in THF(200 mL) was added borane.THF complex (1.0 M, 210 mL, 210 mmol). Themixture was allowed to stir for 24 hours. The reaction was quenched withwater. The solvent THF was removed under reduced pressure. The resultingsolid was dissolved in ethyl acetate (500 mL), washed with 1Nhydrochloric acid, saturated sodium bicarbonate, and brine. The combinedorganic layers were dried over sodium sulfate and concentrated to afford(3-bromo-2-methylphenyl)methanol.

Step B: 5-Bromo-4-methyl-2-benzofuran-1(3H)-one

To a flask charged with (3-bromo-2-methylphenyl)methanol (6.0 g, 30mmol) was added a 1M trifluoroacetic acid solution of thalliumtrifluoroacetate (16.2 g, 29.8 mmol). The mixture was stirred at roomtemperature overnight. The solvent was removed under vacuum, and theresidue was pumped under high vacuum for 30 minutes to ensure completeremoval of TFA. To the residue was then added palladium(II) chloride(529 mg, 2.98 mmol), lithium chloride (2.53 g, 59.7 mmol), magnesiumoxide (2.41 g, 59.7 mmol), and methanol (150 mL). The reaction wasflushed with CO twice, and kept under CO at room temperature. Analysisby LC showed a big product spot within 2 hours. To this solution wasadded ethyl acetate to precipitate the salts. The black solution wasfiltered through a Celite® diatomaceous earth pad, washed with EtOAc,adsorbed onto silica and purified by silica gel chromatography to afford5-bromo-4-methyl-2-benzofuran-1(3H)-one.

¹H-NMR (500 MHz, CDCl₃) δ ppm 7.71 (d, J=8.0 Hz, 1H), 7.58 (d, J=8.0 Hz,1H), 5.25 (s, 2H), 2.37 (s, 3H).

Intermediate 2

4-Methyl-5-oxiran-2-yl-2-benzofuran-1(3H)-one Step A:5-Ethenyl-4-methyl-2-benzofuran-1(3H)-one

5-Bromo-4-methyl-2-benzofuran-1(3H)-one [INTERMEDIATE 1] (600 mg, 4.5mmol), potassium vinyl trifluoroborate (510 mg, 2.2 mmol),PdCl₂(dppf)-CH₂Cl₂ Adduct (180 mg, 0.220 mmol), and TEA (0.62 mL, 4.5mmol) were added to 10 mL ethanol in a 20 mL microwave tube. The tubewas sealed and degassed, then heated to 140° C. for 20 minutes. Analysisby LC-MS showed product peak. The reaction mixture was diluted withethyl acetate, washed with brine twice, dried and evaporated to dryness.The crude product was purified by MPLC chromatography (0-80%ETOAC/Hexane solvent system) to yield5-ethenyl-4-methyl-2-benzofuran-1(3H)-one.

¹H-NMR (500 MHz, CDCl₃): δ ppm 7.76 (d, J=8 Hz, 1H), 7.03 (dd, J=11, 17Hz, 1H), 5.84 (d, J=17 Hz, 1H), 5.55 (d, J=11 Hz, 1H), 5.29 (s, 2H),2.34 (s, 3H). LC-MS: [M+1]=175.

Step B: 4-Methyl-5-oxiran-2-yl-2-benzofuran-1(3H)-one

5-ethenyl-4-methyl-2-benzofuran-1(3H)-one (1.5 g, 8.4 mmol) was added toDCM (25 mL) at 0° C. then meta-chloroperbenzoic acid (2.9 g, 17 mmol)was added and the mixture was stirred at room temperature overnight. Thereaction mixture was washed once each with saturated aqueous Na₂S₂O₃,saturated sodium bicarbonate, and brine. The organic layer was driedover Na₂SO₄, filtered, and evaporated to dryness. The crude material waspurified by MPLC chromatography (eluting with 0-80% EtOAc/hexane solventsystem) to yield 4-methyl-5-oxiran-2-yl-2-benzofuran-1(3H)-one.

¹H-NMR (500 MHz, CDCl₃): δ ppm 7.77 (d, J=8 Hz, 1H), 7.43 (d, J=8 Hz,1H), 5.30 (s, 2H), 4.12 (s, 1H), 3.27 (t, J=4 Hz, 1H), 2.74 (dd, J=2.2,5.5 Hz, 1H), 2.43 (s, 3H).

LC-MS: [M+1]=191.

Intermediates 2A and 2B

2A: 4-Methyl-5-[(2S)-oxiran-2-yl]-2-benzofuran-1(3H)-one 2B:4-Methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one

Racemic 4-methyl-5-oxiran-2-yl-2-benzofuran-1(3H)-one [INTERMEDIATE 2]was resolved on a ChiralPak® AD-H column (5×25 cm) under supercriticalfluid chromatography (SFC) conditions on a Berger MGIII preparative SFCinstrument. The racemate was diluted to 50 mg/ml in 1:1 DCM:MeOH. Theseparation was accomplished using 10% EtOH/CO2, flow rate 200 ml/min,100 bar, 25° C. 500 μl Injections were spaced every 2.12 minutes. Thefast epoxide (4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one, 2B)eluted at 5.2 min, and the slow epoxide(4-methyl-5-[(2S)-oxiran-2-yl]-2-benzofuran-1(3H)-one, 2A) eluted at 5.6minutes.

Alternatively, the resolution could also be achieved using a mobilephase of 8% MeOH/98% CO₂ with a flow rate of 100 ml/min. In that casethe sample was prepared by dissolving in methanol, 20 mg/ml, and using a1 mL volume per injection. After separation, the fractions were driedoff via rotary evaporator at bath temperature 40° C.

The absolute stereochemistry of each enantiomer was inferred based onthe X-ray crystal structure determination of a derivative made with 2B,and by Mosher ester and Trost ester ¹HNMR analysis of an ester madestarting from 2B. The B epoxide isomer finds utility in the presentinvention.

Intermediate 2 Method 2

4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one Step A:3-hydroxymethyl-2-methyl phenol

To a SL 3 neck RB flask equipped with overhead stirrer was charged NaBH4(87.0 g, 2.30 mol) and THF (3.0 L) and the resulting slurry was cooledto 10° C. To the slurry was then added 3-hydroxy-2-methyl benzoic acid(175 g, 1.15 mol) portionwise over 20 minutes (Tmax 17° C.). A stirrableslurry formed, and was aged for an additional 45 minutes at 10-15° C.after which BF₃—OEt₂ (321 mL, 2.53 mol) was added slowly over 1.5 hours.The slurry was aged at 10° C.-15° C. for 2 hours and then assayed forreaction completion (98.5% conversion). The slurry was cooled to <10° C.and quenched with 931 mL MeOH slowly over 1.5 h (gas evolution). Theresulting slurry was aged overnight at room temperature. The batch wascooled to <10° C. then quenched with 1 N HCl (1.5 L) to get ahomogeneous solution (pH solution ˜1), which was aged for 30 minutes.The organic solvents were then removed by rotary evaporation toapproximately 1.8 L of total reaction volume (bath temperature was setto 50° C.; internal temp of concentrate after rotary evaporation was˜40° C.). The slurry was held at 45° C. for 30 minutes and then cooledslowly to 15° C. The solids were filtered and washed with cold (15° C.)water (2×300 mL), providing 3-hydroxymethyl-2-methyl phenol.

¹H-NMR (400 MHz, DMSO-d₆): δ 9.11 (s, 1H), 6.95 (t, J=7.8 Hz, 1H), 6.82(d, J=7.4 Hz, 1H), 6.71 (d, J=7.8 Hz, 1H), 4.93 (t, J=5.5 Hz, 1H), 4.44(d, J=5.5 Hz, 2H), 2.06 (s, 3H).

Step B: 4-Bromo-3-hydroxymethyl-2-methyl phenol

3-Hydroxymethyl-2-methyl phenol (113.9 g, 824.0 mmol) was dissolved in amixture of acetonitrile (850 mL) and trifluoroacetic acid (750.0 mL,9.735 mmol) in a 3-neck 5-L flask under nitrogen. The reaction mixturewas cooled to −33° C. N-bromosuccinimide (141 g, 791 mmol) was addedover 15 minutes, with the temperature during addition in the range of−35 to −33° C. The reaction mixture was allowed to stir for anadditional 15 minutes during which time the temperature decreased to−40° C. The cooling bath was removed, and potassium carbonate (741.0 g,5.358 mmol) diluted with water to a total of 1.0 L was added.Off-gassing was observed, and the temperature increased to 25° C. MTBE(1.5 L) was added, and the reaction mixture was transferred to aseparatory funnel. The layers were separated. The aqueous layer wasdiluted with water (500 mL) and extracted with MTBE (1 L)+EtOAc (500mL), and then MTBE (500 mL)+EtOAc (250 mL). The combined organic layerswere washed with water (240 mL) and dried over sodium sulfate. Thesodium sulfate was removed by filtration, washed with additional MTBEand concentrated under reduced pressure. MTBE (684 mL, 2 volumes) wasadded, and the suspension was heated to 40° C. to produce a homogeneoussolution. The solution was allowed to cool to room temperature. Sixvolumes of heptane were added, and the suspension was stirred overnight.The suspension was filtered, and the crystals were washed with 4:1heptane:MTBE (500 mL), followed by heptane (500 mL). The solid was driedunder vacuum, providing 4-bromo-3-hydroxymethyl-2-methyl phenol.

¹H NMR (400 MHz, DMSO-d₆): δ 9.52 (s, 1H), 7.21 (d, J=8.6 Hz, 1H), 6.71(d, J=8.6 Hz, 1H), 4.88 (t, J=5.1 Hz, 1H), 4.59 (d, J=5.1 Hz, 2H), 2.23(s, 3H)

Step C: 5-Hydroxy-4-methyl-3H-isobenzofuran-1-one

To a 2 L 3 neck flask equipped with overhead stirrer, N₂ inlet, andcondenser were charged 4-bromo-3-hydroxymethyl-2-methyl phenol (100 g,461 mmol), CuCN (83.0 g, 921 mmol), and DMF (500 mL). The solution wassparged with N₂ for 15 minutes and then heated to 145° C. to obtain ahomogeneous solution. The solution was aged at 145° C. for 2 hours, andthen the reaction mixture was cooled to 95° C. 41.5 mL water was added(sparged with N₂), and the reaction aged for 20 hours. The reaction wascooled to room temperature and then the solids filtered throughSolka-Flok® powdered cellulose and the cake washed with 50 mL DMF. To a3 L flask containing 1 L EtOAc was added the DMF filtrate. A precipitatecoating formed in the bottom of the flask. The DMF/EtOAc suspension wasfiltered through Solka Flok® and the cake was washed with 250 mL EtOAc.The resulting filtrate was washed with 5% brine solution (3×500 mL). Theaqueous layers were extracted with 500 mL EtOAc and the combinedorganics were dried over MgSO4, filtered and evaporated. The solids wereslurried in 250 mL MTBE at room temperature and then filtered and washedwith 100 mL MTBE. The solids were dried under vacuum at roomtemperature, providing 5-hydroxy-4-methyl-3H-isobenzofuran-1-one.

¹H NMR (400 MHz, DMSO-d₆): δ 10.52 (s, 1H), 7.51 (d, J=8.3 Hz, 1H), 6.99(d, J=8.3 Hz, 1H), 5.28 (s, 2H), 2.07 (s, 3H).

Step D: Trifluoromethanesulfonic acid4-methyl-1-oxo-1,3-dihydro-isobenzofuran-5-yl ester

5-Hydroxy-4-methyl-3H-isobenzofuran-1-one (46.8 g, 285 mmol) wassuspended in dichloromethane (935 mL) in a 2-L roundbottom flaskequipped with overhead stirrer under nitrogen. Triethylamine (59.5 mL,427 mmol) was added, and the reaction mixture was cooled in an ice bathto 3.8° C. Trifluoromethanesulfonic anhydride (67.4 mL, 399 mmol) wasadded via addition funnel over 50 minutes, keeping the temperature <10°C. After stirring the reaction mixture for an additional 15 minutes, thereaction mixture was quenched with water (200 mL), and then stirred withDARCO® KB (activated carbon, 25 g) for 15 minutes. The biphasic mixturewas filtered over Solka-Floc®, washing with additional dichloromethane,and transferred to a reparatory funnel, whereupon it was diluted withadditional water (300 mL). The layers were separated, and the organiclayer was washed with water (500 mL) and 10% brine (200 mL). Thedichloromethane solution was dried over sodium sulfate, filtered andevaporated. The orange-red solid was adsorbed onto silica gel (27.5 g)and eluted through a pad of silica gel (271 g) with 25% ethylacetate/hexanes. The resulting solution was concentrated under vacuumwith the product crystallizing during concentration. The suspension wasfiltered, and the solid was washed with heptane and dried under vacuumand nitrogen, providing trifluoromethanesulfonic acid4-methyl-1-oxo-1,3-dihydro-isobenzofuran-5-yl ester. ¹H NMR (400 MHz,CDCl₃): δ 7.87 (d, J=8.4 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H), 5.32 (s, 2H),2.41 (s, 3H)

Step E: 5-(1-Butoxy-vinyl)-4-methyl-3H-isobenzofuran-1-one

To a 1 L 3-neck was charged trifluoromethanesulfonic acid,4-methyl-1-oxo-1,3-dihydro-isobenzofuran-5-yl ester (63.0 g, 213 mmol),DMF (315 mL), butyl vinyl ether (138 mL, 1063 mmol)) and then Et₃N (35.6mL, 255 mmol). The solution was sparged with N₂ for 20 minutes. To thesolution was added Pd(OAc)₂ (1.19 g., 5.32 mmol) and DPPP (2.41 g., 5.85mmol). The solution was sparged for an additional 10 minutes and thenheated to 80° C. After a 1 hour age, the solution was cooled to <10° C.,quenched with 630 mL EtOAc, washed with 5% NH₄Cl (2×315 mL) and 10%brine (2×315 mL), dried over MgSO₄, filtered, concentrated by rotaryevaporation and then flushed with EtOAc (3×100 mL) to remove excessbutyl vinyl ether, providing crude5-(1-butoxy-vinyl)-4-methyl-3H-isobenzofuran-1-one.

¹H NMR (400 MHz, DMSO-d₆): δ 7.67 (d, J=7.7 Hz, 1H), 7.48 (d, J=7.7 Hz,1H), 5.42 (s, 2H), 4.54 (d, J=2.3 Hz, 1H), 4.27 (d, J=2.3 Hz, 1H), 3.85(t, J=6.4 Hz, 2H), 2.27 (s, 3H), 1.71-1.64 (m, 2H), 1.46-1.37 (m, 2H),0.92 (t, J=7.4 Hz, 3H)

Step F: 5-(2-Bromo-acetyl)-4-methyl-3H-isobenzofuran-1-one

To a 1 L 3-neck flask equipped with overhead stirrer was added crude5-(1-butoxy-vinyl)-4-methyl-3H-isobenzofuran-1-one (55.8 g) and THF (315mL). The solution was cooled to <5° C. after which water (79 mL) wasadded and the solution was maintained at <5° C. NBS (41.6 g) was thenadded portionwise while maintaining Tmax=19° C. The solution was thenwarmed to room temperature for 30 minutes. HBr (48%, 0.241 mL) was addedand the reaction was aged at room temperature for approximately 1 hourafter which 236 mL water was then added to the batch. A water bath isused to maintain the temperature at 20° C. Another 315 mL of water wasadded (solvent composition 1:2 THF:water) and the slurry was cooled to15° C. The resulting solids were filtered and washed with cold 1:2THF:water (15° C.): 150 mL displacement wash followed by 100 mL slurrywash. The solids were dried under vacuum at room temperature to provide5-(2-bromo-acetyl)-4-methyl-3H-isobenzofuran-1-one.

¹H NMR (400 MHz, DMSO-d₆): δ 7.99 (d, J=7.8 Hz, 1H), 7.82 (d, J=7.8 Hz,1H), 5.49 (s, 2H), 4.92 (s, 2H), 2.33 (s, 3H)

Step G: 4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one

5-(2-Bromo-acetyl)-4-methyl-3H-isobenzofuran-1-one (48.8 g., 181 mmol)was charged to a 5 L 3 neck round bottom equipped with overhead stirrer,thermocouple, and heating mantle. 2-Propanol (1.22 L) was added,followed by 610 mL of pH 7 0.1M potassium phosphate buffer. Buffersolution (610 mL) was charged to a 1.0 L Erlenmeyer flask, and 2.44 g ofNADP was added to the Erlenmeyer and swirled to dissolve. A reducingenzyme, KRED MIF-20 (2.44 g) (available from Codexis, Inc., 200Penobscot Drive, Redwood City, Calif. 94063, www.codexis.com, tel.1-650-421-8100) was added to the Erlenmeyer flask and the mixture wasswirled to dissolve the solids. The resulting solution was added to the5 L round bottom, which was then heated to 28° C. and aged for 6 hours,at which point the reaction was cooled to room temperature andtriethylamine (50.2 mL, 360 mmol) was added. The resulting solution wasaged at 40° C. for 1 hour. The light slurry solution was cooled to roomtemperature, after which 122 g NaCl was added. The solution was aged atroom temperature and then extracted with 1.22 L isopropyl acetate(IPAc). The aqueous layer was re-extracted with 400 mL IPAc and thecombined organics were washed with 400 mL 20% brine solution, dried overMgSO₄, filtered and concentrated by rotary evaporation. The resultingsolids were taken up in 100 mL IPAc (thick slurry). Hexanes were added(400 mL) and the suspension aged at room temperature and then filteredand washed w/5:1 Hexanes:IPAc solution (150 mL). The crystalline solidswere dried under vacuum at room temperature to provide4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one.

¹H NMR (400 MHz, CDCl₃): δ 7.75 (d, J=8.1 Hz, 1H), 7.42 (d, J=8.1 Hz,1H), 5.28 (s, 2H), 4.10 (dd, J=4.0, 2.8, 1H), 3.26 (dd, J=5.6, 4.0, 1H),2.72 (dd, J=5.6, 2.8, 1H), 2.42 (s, 3H).

Intermediate 3

5-{(1R)-2-(2,7-Diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl)-4-methyl-2-benzofuran-1(3H)-onehydrochloride Step A: tert-Butyl2-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2,7-diazaspiro[4.5]decane-7-carboxylate

A solution of 4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one(INTERMEDIATE 2) (38 mg, 0.20 mmol) in 2 mL ethanol was added totert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate (40 mg, 0.20 mmol).The reaction mixture was microwaved at 140° C. for 55 minutes. Thesolvents were removed in vacuo to provide tert-butyl2-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2,7-diazaspiro[4.5]decane-7-carboxylatewhich was carried on without further purification.

LC-MS (IE, m/z): 431 [M+1]⁺.

Step B:5-{(1R)-2-(2,7-Diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride

A suspension of tert-butyl24(R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2,7-diazaspiro[4.5]decane-7-carboxylate(80 mg, 0.20 mmol) in dioxane (200 uL) was treated with a solution ofhydrochloric acid in dioxane (4.0 M, 200 μL). After shaking for 3 hours,the solution was treated with additional hydrochloric acid in dioxane(4.0 M, 100 μL). After shaking an additional sixteen hours, the solventswere removed in vacuo to provide5-{(1R)-2-(2,7-diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride which was carried on without further purification.

LC-MS (IE, m/z): 331 [M+1]⁺.

Intermediate 4

5-{(1R)-2-(2,8-Diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride

5-{(1R)-2-(2,8-Diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from commercially availabletert-butyl 2,8-diazaspiro[4.5]decane-7-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 331 [M+1]⁺.

Intermediate 5

5-{(1R)-1-Hydroxy-2-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)ethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride

5-{(1R)-1-Hydroxy-2-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)ethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyloctahydro-5H-pyrrolo[3,4-c]pyridine-5-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 317 [M+1]⁺.

Intermediate 6

5-[(R)-2-(Hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride

5-[(R)-2-(Hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-bezofuran1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyl3,3-dimethylpiperazine-1-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 289 [M+1]⁺.

Intermediate 7

5-[(R)-2-(1,7-Diazaspiro[4.5]dec-7-yl)-1-hydroxethyl]-4-benzofuran1(3H)-onehydrochloride

5-[(R)-2-(1,7-Diazaspiro[4.5]dec-7-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyl1,7-diazaspiro[4.5]decane-1-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 331 [M+1]⁺.

Intermediate 8

5-[(R)-2-(2,8-Diazaspiro[5.5]undec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride

5-[(R)-2-(2,8-Diazaspiro[5.5]undec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyl2,8-diazaspiro[5.5]undecane-2-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 345 [M+1]⁺.

Intermediate 9

5-((1R)-2-(Hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride

5-((1R)-2-(Hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butylhexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 289 [M+1]⁺.

Intermediate 10

4-Methoxy-6-(oxiran-2-yl)pyridine-3-carbonitrile Step A:5-Bromo-2-chloro-4-methoxypyridine

To a solution of 2-chloro-4-methoxypyridine (10.0 g, 69.7 mmol) in 50 mLof sulfuric acid at 0° C. was added NBS. The reaction mixture wasallowed to stir and warm up to room temperature for 2 hours and thenheated at 60° C. for 5 hours. The reaction mixture was then cooled toroom temperature and neutralized with 1 N NaOH (pH ˜7), diluted withwater (50 mL) and the aqueous layer was extracted with ethyl acetate(2×100 mL). The organic layers were washed with water (2×50 mL),saturated NaHCO₃ and brine, dried over Mg₂SO₄ and concentrated toprovide an oil, which was chromatographed. On elution with 0-25%EtOAc/hexanes, the final product was obtained.

¹H NMR (500 MHz, DMSO-d₆), δ 8.4 (s, 1H), 7.29 (s, 1H), 3.97 (s, 3H);

LC/MS (M+1)⁺=223.

Step B: 6-Chloro-4-methoxypyridine-3-carbonitrile

A solution of 5-bromo-2-chloro-4-methoxypyridine (5.0 g, 22.48 mmol) inDMF (80 mL) was purged with nitrogen for 15 minutes. At this point,Zn(CN)₂ (3.96 g, 33.7 mmol) and Pd(Ph₃P)₄ (2.60 g, 2.25 mmol) wereadded, successively. The resulting suspension was stirred at 95° C. for12 hours under nitrogen atmosphere. The reaction mixture was cooled toambient temperature, and filtered to remove inorganic solid. The solvent(DMF) was evaporated to provide the crude residue as an oil, which waspurified on silica gel and eluted with 0-30% ethyl acetate/hexanes toafford the product.

¹H NMR (500 MHz, DMSO-d₆), δ 8.69 (s, 1H), 7.50 (s, 1H), 4.04 (s, 3H);LC/MS (M+1)⁺=169.

Step C: 6-Ethenyl-4-methoxypyridine-3-carbonitrile

A 20 ml, microwave tube was charged with6-chloro-4-methoxypyridine-3-carbonitrile (200.0 mg, 1.2 mmol),bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex withdichloromethane (97.0 mg, 0.12 mmol), potassium vinyl trifluoroborate(318.0 mg, 2.37 mmol), triethylamine (0.33 mL, 2.37 mmol), and EtOH (6mL). The microwave tube was evacuated and filled with nitrogen (twotimes) and heated to 140° C. After 1 hour, the reaction mixture wasdiluted with water and extracted with EtOAc. The combined organic layerswere washed with brine and dried over Na₂SO₄. The extracts wereconcentrated and chromatographed over a column of SiO₂ (0-30%EtOAc/hexanes as eluent). Evaporation of the solvent yielded the titlecompound.

¹H NMR (500 MHz, DMSO-d₆), δ 8.65 (s, 1H), 6.89 (s, 1H), 6.83 (dd,J=10.7 Hz, 1H), 6.42 (d, J=7.3 Hz, 1H), 5.70 (d, J=10.6 Hz, 1H) 4.05 (s,3H); LC/MS (M+1)⁺=161.

Step D: 6-(2-Bromo-1-hydroxyethyl)-4-methoxypyridine-3-carbonitrile

A solution of 6-ethenyl-4-methoxypyridine-3-carbonitrile (80.0 mg, 0.499mmol) in 1,4-dioxane (8 mL) and H₂O (4 mL) was treated withN-bromosuccinimide (89.0 mg, 0.499 mmol, 1.0 equiv). The reactionmixture was allowed to stir at room temperature overnight. The reactionmixture was poured into H₂O (8 mL) and extracted with EtOAc (3×30 mL).The combined organic layers were washed with saturated aqueous NaCl(1×30 mL), and dried over Na₂SO₄. Evaporation of the solvent gave an oilthat was purified over SiO₂ (0-30% EtOAc/hexanes as eluent) yielding6-(2-bromo-1-hydroxyethyl)-4-methoxypyridine-3-carbonitrile. ¹HNMR (500MHz, DMSO-d₆), δ 8.65 (s, 1H), 7.19 (s, 1H), 5.05 (t, J=5.4 Hz, 1H),4.05 (s, 3H), 3.85 (dd, J=4.5 Hz, 1H), 3.75 (dd, J=6.1 Hz, 1H); LC/MS(M+1)⁺=241.

Step E: 4-Methoxy-6-(oxiran-2-yl)pyridine-3-carbonitrile

A solution of6-(2-bromo-1-hydroxyethyl)-4-methoxypyridine-3-carbonitrile (74.0 mg,0.288 mmol) in anhydrous methanol (7 mL) was treated with sodiumcarbonate (61.0 mg, 0.576 mmol, 2.0 equiv), and allowed to stir at roomtemperature overnight. The solvent was evaporated. The residue was takenup in EtOAc (30 mL) and washed with water and brine. After drying overNa₂SO₄, the organic layer was removed and the residue was purified overSiO₂ (10-45% EtOAc/hexanes as eluent) to yield4-methoxy-6-(oxiran-2-yl)pyridine-3-carbonitrile. ¹H NMR (500 MHz,DMSO-d₆), δ 8.64 (s, 1H), 6.87 (s, 1H), 4.08 (dd, J=2.6 Hz, J=2.3 Hz,1H), 4.03 (s, 3H), 3.26 (dd, J=4.6 Hz, J=5.4 Hz, 1H), 2.87 (dd, J=2.2Hz, J=2.4 Hz, 1H); LC/MS (M+1)⁺=177.

Resolution of the epoxides was carried out (prep SFC, 160 mL/min., 10%MeOH in SC CO₂, AD-H) to provide:

Isomer A: (M+1)⁺=177.

Isomer B: (M+1)⁺=177.

Intermediate 11

6-(Oxiran-2-yl)pyridine-3-carbonitrile Step A:6-Ethenylpyridine-3-carbonitrile

To a stirring solution of 6-bromopyridine-3-carbonitrile (2.0 g, 10.9mmol), in EtOH (70 mL) were addedbis[(diphenylphosphino)ferrocene]dichloropalladium(II), complex withdichloromethane (0.892 mg, 0.10 mmol), potassium vinyl trifluoroborate(2.93 g, 21.9 mmol), triethylamine (3.0 mL, 21.9 mmol), and water (0.5mL). The reaction mixture was heated to reflux. Upon completion asdetermined by reverse phase HPLC-MS (1-2 h) and TLC (eluent: 10% ethylacetate in hexanes), the reaction was cooled to room temperature, andthen was diluted with water and extracted with EtOAc. The combinedorganic layers were washed with brine and dried over MgSO₄. The extractswere concentrated and chromatographed over a column of SiO₂ (0-20%EtOAc/hexanes as eluent). Evaporation of the solvent yielded6-ethenylpyridine-3-carbonitrile.

¹H NMR (500 MHz, CDCl₃), δ 8.85 (s, 1H), 7.94-7.93 (m, 1H), 6.89-6.83(m, 1H), 7.45 (d, J=8.2 Hz, 1H), 6.85 (dd, J=10.8, Hz, 1H), 6.42 (d,J=17.4 Hz, 1H); LC/MS (M+1)⁺=131.

Step B: 6-(Oxiran-2-yl)pyridine-3-carbonitrile

A solution of 6-ethenylpyridine-3-carbonitrile (0.742 g, 5.70 mmol) in a2:1 ratio of H₂O:t-BuOH (30 mL) was treated with N-bromosuccinimide inportions over 5 minutes (1.07 g, 5.99 mmol) and stirred at 40° C. for 1hour. After cooling to 5° C., the reaction was basified with drop wiseaddition of solution of sodium hydroxide (0.684 g in 5 mL of H₂O, 17.1mmol) and stirred for another 1 hour. The reaction mixture was pouredinto H₂O (10 mL) and extracted with EtOAc (2×50 mL). The combinedorganic layers were washed with saturated aqueous NaCl (1×30 mL) anddried over MgSO₄. Evaporation of the solvent and purification over SiO₂(0-30% EtOAc/hexanes as eluent) provided6-(oxiran-2-yl)pyridine-3-carbonitrile.

¹H NMR (500 MHz, CDCl₃), δ 8.87 (s, 1H), 7.99 (d, J=8.1 Hz, 1H), 7.40(d, J=8.1 Hz, 1H), 4.11 (s, 1H), 4.08 (dd, J=2.6 Hz, J=2.3 Hz, 1H), 3.29(m, 1H), 2.94 (m, 1H); LC/MS (M+1)⁺=147.

Resolution of the epoxides was carried out (prep SFC, 160 mL/min., 10%MeOH in SC CO₂, AD-H) to provide:

Isomer A: (M+1)⁺=147.

Isomer B: (M+1)⁺=147.

Intermediate 12

5-((1R)-1-Hydroxy-2-(1,7-diazaspiro[4.4]nonan-1-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride

5-((1R)-1-Hydroxy-2-(1,7-diazaspiro[4.4]nonan-1-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyl1,7-diazaspiro[4.4]nonane-7-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 317 [M+1]⁺.

Intermediate 13

5-((1R)-1-Hydroxy-2-(2-methylhexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride

5-((1R)-1-Hydroxy-2-(2-methylhexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyl2-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 317 [M+1]⁺.

Intermediate 14

6-(1H-Tetrazol-1-yl)nicotinic acid Step A: Methyl6-(1H-tetrazol-1-yl)nicotinate

A mixture of methyl 6-aminonicotinate (5.0 g, 33 mmol) in acetic acid(47 ml, 820 mmol) was treated with triethyl orthoformate (8.8 ml, 53mmol), followed by sodium azide (3.2 g, 49 mmol). The resulting mixturewas heated at 80° C. for 1 hour, after which the reaction mixture wascooled to room temperature and diluted with water. The resultingprecipitate was collected and dried under high vacuum to provide methyl6-(1H-tetrazol-1-yl)nicotinate.

LC-MS (IE, m/z): 206 [M+1]⁺.

Step B: 6-(1H-Tetrazol-1-yl)nicotinic acid

Methyl 6-(1H-tetrazol-1-yl)nicotinate was dissolved in THF (50 mL) andtreated with 1N lithium hydroxide (50 mL) and stirred for 1 hour. Themixture was diluted with water and the resulting solid isolated byfiltration and drying under high vacuum to provide6-(1H-tetrazol-1-yl)nicotinic acid.

¹H-NMR (CDCl₃, 500 MHz), δ 9.63 (s, 1H), 9.19 (s, 1H), 8.62 (m, 1H),8.22 (m, 1H), 4.04 (s, 3H); LC-MS (IE, m/z): 192 [M+1]⁺.

Intermediate 15

5-(1H-Tetrazol-1-yl)picolinic acid

5-(1H-tetrazol-1-yl)picolinic acid was prepared in a similar fashion tothat described for the synthesis of INTERMEDIATE 14 starting from methyl6-aminonicotinate.

LC-MS (IE, m/z): 192 [M+1]⁺.

Intermediate 16

5-((1R)-1-Hydroxy-2-(1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride

5-((1R)-1-Hydroxy-2-(1,7-diazaspiro[4.4]nonan-1-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from tert-butyl1,7-diazaspiro[4.4]nonane-1-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 317 [M+1]⁺.

Intermediate 17 A and B

5-((1R)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-oneHydrochloride

5-((1R)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from racemic (cis)-tert-butyl3,6-diazabicyclo[3.2.0]heptane-6-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].Following epoxide coupling, the diastereomers were separated by chiralSFC and the individual isomers deprotected.

Faster eluting; LC-MS (IE, m/z): 289 [M+1]⁺.

Slower eluting; LC-MS (IE, m/z): 289 [M+1]⁺.

Intermediate 18 A and B

6-((1S)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methoxynicotinonitrilehydrochloride

6-((1S)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methoxynicotinonitrilehydrochloride was prepared in a similar fashion to that described forthe synthesis of INTERMEDIATE 3 starting from racemic (cis)-tert-butyl3,6-diazabicyclo[3.2.0]heptane-6-carboxylate and the slower elutingisomer of 4-methoxy-6-(oxiran-2-yl)pyridine-3-carbonitrile (INTERMEDIATE10 A). Following epoxide coupling, the diastereomers were separated bychiral SFC and the individual isomers deprotected.

Faster eluting; LC-MS (IE, m/z): 275 [M+1]⁺.

Slower eluting; LC-MS (IE, m/z): 275 [M+1]⁺.

Example 1

5,5′-{2,7-Diazaspiro[4.5]decane-2,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)

A solution of 4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one(INTERMEDIATE 2) (38 mg, 0.20 mmol) in 0.50 mL of ethanol was prepared.Separately,5-{(1R)-2-(2,7-diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-one(INTERMEDIATE 3) (60 mg, 0.20 mmol) was dissolved in 1 mL of ethanol byaddition of 200 mg of MP-CO3 resin and heating. The two solutions, alongwith the resin, were combined and microwaved at 140° C. for fifty-fiveminutes. The solvent was removed in vacuo and the remaining solids weredissolved in 1 mL DMSO.

Purification by HPLC afforded5,5′-{2,7-Diazaspiro[4.5]decane-2,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one).

LC-MS (IE, m/z): 521 [M+1]⁺.

Example 2

5,5′-{2,8-diazaspiro[4.5]decane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)

5,5′-{2,8-diazaspiro[4.5]decane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)was prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting from5-{(1R)-2-(2,8-Diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 4) and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 467 [M+1]⁺.

Example 3

6-({2-[(2R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]octahydro-5H-pyrrolo[3,4-c]pyridine-5-yl}carbonyl)pyridine-3-carbonitrile

A solution of 5-cyanopyridine-2-carboxylic acid (25 mg, 0.12 mmol) andTBTU (64 mg, 0.20 mmol) in 0.5 mL of DMF was added to5-{(1R)-1-hydroxy-2-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)ethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 5) (30 mg, 0.10 mmol). DIEA (87 uL, 0.50mmol) was added and the solution was shaken for sixteen hours and thendiluted with 0.5 mL dimethylsulfoxide. The resulting solution waspurified by HPLC which afforded6-({2-[(2R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]octahydro-5H-pyrrolo[3,4-c]pyridine-5-yl}carbonyl)pyridine-3-carbonitrile.

LC-MS (IE, m/z): 447 [M+1]⁺.

Example 4A and B

5,5′-{Hexahydropyrrolo[3,4-b]pyrrole-1,5-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one

A solution of 4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one[INTERMEDIATE 2B] (38 mg, 0.20 mmol) in 0.50 mL of ethanol was prepared.Separately,5-[(R)-2-(hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-bezofuran1(3H)-onehydrochloride [INTERMEDIATE 6] (60. mg, 0.20 mmol) was dissolved in 1 mLof ethanol by addition of 200 mg of MP-CO3 resin and heating. The twosolutions, along with the resin, were combined and heated in a microwavereactor at 140° C. for fifty-five minutes. The resulting mixture wasconcentrated in vacuo and the remaining solids were dissolved in 1 mLDMSO.

Purification of the DMSO solution by HPLC afforded two diastereomers of5,5′-{hexahydropyrrolo[3,4-b]pyrrole-1,5-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one:

Faster eluting; LC-MS (IE, m/z): 493 [M+1]⁺.

Slower eluting; LC-MS (IE, m/z): 493 [M+1]⁺.

Example 5

5,5′-{1,7-Diazaspiro[4.5]decane-1,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)

5,5′-{1,7-Diazaspiro[4.5]decane-1,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)was prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting from5-[(R)-2-(1,7-diazaspiro[4.5]dec-7-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride [INTERMEDIATE 7] and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 521 [M+1]⁺.

Example 6

5,5′-{2,8-Diazaspiro[5.5]undecane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)

5,5′-{2,8-Diazaspiro[5.5]undecane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one)was prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting from5-[(R)-2-(2,8-Diazaspiro[5.5]undec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride (INTERMEDIATE 8) and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 535 [M+1]⁺.

Example 7A and B

6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrile

6-(1-Hydroxy-2-{5-[(2R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl}ethyl)pyridine-3-carbonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 4A and B starting from5-[(R)-2-(hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-bezofuran1(3H)-onehydrochloride [INTERMEDIATE 6] and the slower eluting diastereomer of6-(oxiran-2-yl) pyridine-3-carbonitrile [INTERMEDIATE 11 B].

Faster eluting diastereomer: LC-MS (IE, m/z): 449 [M+1]⁺.

Slower eluting diastereomer: LC-MS (IE, m/z): 449 [M+1]⁺.

Example 8A and B

4-(1-((R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-5(1H)-ylsulfonyl)benzonitrile

A solution of commercially available 4-cyanobenzene-1-sulfonyl chloride(31 mg, 0.12 mmol) in 0.3 mL of DMF was added to a solution of5-[(1R)-2-(2,8-diazaspiro[5.5]undec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 6) in 0.2 mL of DMF and DIEA (87 uL, 0.50mmol). The combined solution was shaken for sixteen hours and thendiluted with 0.5 mL DMSO. The resulting solution was purified by HPLC toafford the two diastereomers of4-(1-((R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-5(1H)-ylsulfonyl)benzonitrile

Faster eluting diastereomer: LC-MS (IE, m/z): 454 [M+1]⁺.

Slower eluting diastereomer: LC-MS (IE, m/z): 454 [M+1]⁺.

Example 9

4-(5-((R)-2-Hydroxy-2-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-ylsulfonyl)benzonitrile

4-(5-((R)-2-Hydroxy-2-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-ylsulfonyl)benzonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 8 starting from5-((1R)-2-(hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride [INTERMEDIATE 9] and 4-cyanobenzene-1-sulfonyl chloride.

LC-MS (IE, m/z): 454 [M+1]⁺.

Example 10A and B

5,5′-(1R,1′R)-2,2′-(1,7-Diazaspiro[4.4]nonane-1,7-diyl)bis(1-hydroxyethane-2,1-diyl)bis(4-methylisobenzofuran-1(3H)-one)

5,5′-(1R,1′R)-2,2′-(1,7-Diazaspiro[4.4]nonane-1,7-diyl)bis(1-hydroxyethane-2,1-diyl)bis(4-methylisobenzofuran-1(3H)-one)was prepared in a similar fashion to that described for the synthesis ofEXAMPLE 4A and B starting from tert-butyl1,7-diazaspiro[4.4]nonane-7-carboxylate and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

Faster eluting diastereomer: LC-MS (IE, m/z): 508 [M+1]⁺.

Slower eluting diastereomer: LC-MS (IE, m/z): 508 [M+1]⁺.

Example 11A and B

6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrile

6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 4A and B starting from5-((1R)-1-hydroxy-2-(2-methylhexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride [INTERMEDIATE 13] and the slower eluting diastereomer of6-(oxiran-2-yl)pyridine-3-carbonitrile (INTERMEDIATE 11).

Faster eluting diastereomer: LC-MS (IE, m/z): 464 [M+1]⁺.

Slower eluting diastereomer: LC-MS (IE, m/z): 464 [M+1]⁺.

Example 12A and B

5,5′-(1R,1′R)-2,2′-(2-Methylhexahydropyrrolo[3,4-b]pyrrole-1,5-diyl)bis(1-hydroxyethane-2,1-diyl)bis(4-methylisobenzofuran-1(3H)-one)

5,5′-(1R,1′R)-2,2′-(2-Methylhexahydropyrrolo[3,4-b]pyrrole-1,5-diyl)bis(1-hydroxyethane-2,1-diyl)bis(4-methylisobenzofuran-1(3H)-one)was prepared in a similar fashion to that described for the synthesis ofEXAMPLE 4A and B starting from5-((1R)-1-hydroxy-2-(2-methylhexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride [INTERMEDIATE 13] and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

Faster eluting diastereomer: LC-MS (IE, m/z): 507 [M+1]⁺.

Slower eluting diastereomer: LC-MS (IE, m/z): 507 [M+1]⁺.

Example 13A and B

5-((1R)-2-(8-(6-(1H-Tetrazol-1-yl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-2-(8-(6-(1H-Tetrazol-1-yl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 3 starting from5-[(R)-2-(2,8-diazaspiro[5.5]undec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride [INTERMEDIATE 8] and 6-(1H-tetrazol-1-yl)nicotinic acid[INTERMEDIATE 14].

Faster eluting diastereomer: LC-MS (IE, m/z): 518 [M+1]⁺.

Slower eluting diastereomer: LC-MS (IE, m/z): 518 [M+1]⁺.

Example 14

5-((1R)-2-(7-(6-(1H-tetrazol-1-yl)nicotinoyl)-2,7-diazaspiro[4.5]decan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-2-(7-(6-(1H-Tetrazol-1-yl)nicotinoyl)-2,7-diazaspiro[4.5]decan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 3 starting from5-{(1R)-2-(2,7-diazaspiro[4.5]dec-2-yl)-1-hydroxyethyl}-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 3) and 6-(1H-tetrazol-1-yl)nicotinic acid[INTERMEDIATE 14].

LC-MS (IE, m/z): 504 [M+1]⁺.

Example 15

5-((1R)-2-(5-(6-(1H-Tetrazol-1-yl)nicotinoyl)-1H-pyrrolo[3,4-c]_yridine-2(3H,3aH,4H,5H,6H,7H,7aH)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-2-(5-(6-(1H-Tetrazol-1-yl)nicotinoyl)-1H-pyrrolo[3,4-c]_yridine-2(3H,3aH,4H,5H,6H,7H,7aH)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 13 starting from5-{(1R)-1-hydroxy-2-(octahydro-2H-pyrrolo[3,4-c]pyridine-2-yl)ethyl}-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 5) and 6-(1H-tetrazol-1-yl)nicotinic acid[INTERMEDIATE 14].

LC-MS (IE, m/z): 490 [M+1]⁺.

Example 16

5-((1R)-2-(5-(5-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-2-(5-(5-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 13 starting from5-[(1R)-2-(hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 6) and 5-(1H-tetrazol-1-yl)nicotinic acid(INTERMEDIATE 15).

LC-MS (IE, m/z): 490 [M+1]⁺.

LC-MS (IE, m/z): 476 [M+1]⁺.

Example 17

5-((1R)-2-(5-(6-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo-[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-2-(5-(6-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 13 starting from5-[(1R)-2-(hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 6) and 6-(1H-tetrazol-1-yl)nicotinic acid[INTERMEDIATE 14].

LC-MS (IE, m/z): 490 [M+1]⁺.

Example 18A and B

5-((1R)-1-Hydroxy-2-(1-(3-methyl-4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-1-Hydroxy-2-(1-(3-methyl-4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 8 starting from5-((1R)-1-hydroxy-2-(1,7-diazaspiro[4.4]nonan-1-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride [INTERMEDIATE 16] and commercially available3-methyl-4-(1H-tetrazol-1-yl)benzene-1-sulfonyl chloride.

Faster eluting diastereomer (55 A): LC-MS (IE, m/z): 539 [M+1]⁺.

Slower eluting diastereomer (55 B): LC-MS (IE, m/z): 539 [M+1]⁺.

Example 19A and B

5-((1R)-1-Hydroxy-2-(1-(4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-1-Hydroxy-2-(1-(4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 8 starting from5-((1R)-1-hydroxy-2-(1,7-diazaspiro[4.4]nonan-1-yl)ethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride [INTERMEDIATE 16] and commercially available4-(1H-tetrazol-1-yl)benzene-1-sulfonyl chloride.

Faster eluting diastereomer (57 A): LC-MS (IE, m/z): 525 [M+1]⁺.

Slower eluting diastereomer (57 B): LC-MS (IE, m/z): 525 [M+1]⁺.

Example 20

5-((1R)-2-(5-(4-(1H-Tetrazol-1-yl)phenylsulfonyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-2-(5-(4-(1H-Tetrazol-1-yl)phenylsulfonyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 8 starting from5-[(1R)-2-(hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran-1(3H)-onehydrochloride (INTERMEDIATE 6) and commercially available4-(1H-tetrazol-1-yl)benzene-1-sulfonyl chloride.

LC-MS (IE, m/z): 511 [M+1]⁺.

Example 21

5-((1R)-1-Hydroxy-2-(8-(6-(methylsulfonyl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)ethyl)-4-methylisobenzofuran-1(3H)-one

5-((1R)-1-Hydroxy-2-(8-(6-(methylsulfonyl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)ethyl)-4-methylisobenzofuran-1(3H)-onewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 13 starting from5-[(R)-2-(2,8-diazaspiro[5.5]undec-2-yl)-1-hydroxyethyl]-4-methyl-2-benzofuran1(3H)-onehydrochloride [INTERMEDIATE 8] and 6-(methylsulfonyl)nicotinic acid.

LC-MS (IE, m/z): 528 [M+1]⁺.

Slower eluting isomer: LCMS: m/z 523 (M+H)⁺.

Example 22

6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrile

6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting from the faster eluting isomer of5-((1R)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride (INTERMEDIATE 17 A) and the slower eluting diastereomer of6-(oxiran-2-yl)pyridine-3-carbonitrile (INTERMEDIATE 11).

LCMS: m/z 435 (M+H)⁺.

Example 23 Diastereomer of Example 22

6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrile

6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting from the slower eluting isomer of5-((1R)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-onehydrochloride (INTERMEDIATE 17 B) and the slower eluting diastereomer of6-(oxiran-2-yl)pyridine-3-carbonitrile (INTERMEDIATE 11).

LCMS: m/z 435 (M+H)⁺.

Example 24

6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrile

6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting from the faster eluting isomer6-((1S)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methoxynicotinonitrilehydrochloride (INTERMEDIATE 18 A) and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 465 [M+1]⁺.

Example 25 Diastereomer of Example 24

6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrile

6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrilewas prepared in a similar fashion to that described for the synthesis ofEXAMPLE 1 starting the slower eluting isomer6-((1S)-2-(3,6-Diazabicyclo[3.2.0]heptan-3-yl)-1-hydroxyethyl)-4-methoxynicotinonitrilehydrochloride (INTERMEDIATE 18 B) and4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1(3H)-one [INTERMEDIATE 2B].

LC-MS (IE, m/z): 465 [M+1]⁺.

Several assays may be used to measure functional inhibition of the ROMKchannel by compounds of the instant invention. One assay that can beused is an electrophysiology assay that measures the electrical currentthat is generated as potassium permeates through the channel. AnotherROMK functional assay makes use of the ability of thallium to permeatethrough open ROMK channels and increase the fluorescence of a dyepreviously loaded into the cells. Under control conditions, cells loadedwith dye and exposed to thallium-containing medium display atime-dependent increase in fluorescence, the rate of which depends onnumber of functional channels. When cells are incubated in the presenceof a channel inhibitor, the increase in fluorescence is attenuated in aconcentration-dependent manner, and IC₅₀ values of inhibition bycompounds can be accurately determined. This assay has been establishedwith cell lines expressing either human, or rat ROMK channels, andoperates in 384-well format.

Electrophysiology Assay

Block of Kir1.1 (ROMK1) currents was examined by whole cell voltageclamp (Hamill et. al. Pfluegers Archives 391:85-100 (1981)) using theIonWorks® Quattro automated electrophysiology platform (MolecularDevices, Sunnyvale, Calif.). Chinese hamster ovary cells stablyexpressing Kir1.1 channels were maintained in T-75 flasks in cellculture media in a humidified 10% CO₂ incubator at 37° C. Prior to anexperiment, Kir1.1 expression was induced by overnight incubation with 1mM sodium butyrate. On the day of the experiment, cells were dissociatedwith 2.5 ml of Versene™ (Invitrogen 15040-066) for approximately 6minutes at 37° C. and suspended in 10 ml of bath solution containing (inmM): 150 NaCl, 10 KCl, 2.7 CaCl₂, 0.5 MgCl₂, 5 HEPES, pH 7.4. Aftercentrifugation, the cell pellet was resuspended in approximately 4.0 mlof bath solution and placed in the IonWorks® instrument. Theintracellular solution consisted of (in mM): 80 K gluconate, 40 KCl, 20KF, 3.2 MgCl₂, 3 EGTA, 5 Hepes, pH 7.4. Electrical access to thecytoplasm was achieved by perforation in 0.13 mg/ml amphotericin B for 4minutes. Amphotericin B (Sigma A-4888) was prepared as a 40 mg/mlsolution in DMSO.

Voltage protocols and current recordings were performed using theIonWorks® HT software/hardware system. Currents were sampled at 1 kHz.No correction for liquid junction potentials was used. The test pulse,consisting of a 100 ms step to 0 mV from a holding potential of −70 mV,followed by a 100 ms voltage ramp from −70 mV to +70 mV, was appliedbefore and after a 6 minutes compound incubation period. Test compoundswere prepared by diluting DMSO stock solutions into the bath solution at3× the final concentration and placed in the instrument in 96-wellpolypropylene plates. Current amplitudes were measured using theIonWorks® software. To assess compound potency, the fractional blockduring the voltage step to 0 mV was calculated in Microsoft® Excel(Microsoft, Redmond, Calif.), and dose-response curves were fitted withIgor Pro 4.0 (WaveMetrics, Lake Oswego, Oreg.). Normally, a controlcompound is included to support that the assay is giving consistentresults compared to previous measurements, although the control is notrequired to obtain the results for the test compounds. The control was acompound of Formula I of the present invention, preferably with an IC₅₀potency of less than 1 μM in this assay, or another compound (outsidethe scope of Formula I) that has an IC₅₀ potency in this assay of lessthan 1 μM.

Compounds of the Examples were tested in the electrophysiology assay andfound to have a therapeutic level of potency.

Thallium Flux Assay Cell Culture Conditions—

HEK293 cells stably expressing hROMK (hK_(ir)1.1) were grown at 37° C.in a 10% CO₂ humidified incubator in complete growth media: Dulbecco'sModified Eagle Medium supplemented with non-essential amino acids,Penicillin/Streptomycin/Glutamine, G418 and FBS. At >80% confluency, themedia was aspirated from the flask and the sample was rinsed with 10 mlCalcium/Magnesium-free PBS. 5 ml of 1× trypsin (prepared in Ca/Mg FreePBS) was added to a T-225 flask and the flask was returned to 37° C./CO₂incubator for 2-3 minutes. To dislodge the cell, the side of the flaskwas gently banged with a hand. The cells were triturated completely andthen transferred to 25 ml complete media. The sample was thencentrifuged at 1,500 rpm for 6 minutes, followed by resuspension incomplete growth media and then cell concentration was determined. Fortypical re-seeding, 4E6 cells/T-225 flask were found to attain >80%confluency in 4 days. Under ideal growth conditions and appropriatetissue culture practices, this cell line is stable for 40-45 passages.

FluxOR Kit Components (Invitrogen F10017)

-   -   FluxOR™ Reagent (Component A)    -   FluxOR™ Assay Buffer (Component B)—10× Concentrate    -   PowerLoad™ Concentrate (Component C)—100× Concentrate    -   Probenecid (Component D)—Lyophilized sample is kept at −20° C.        Water soluble, 100× after solubilization in 1 ml water. Store at        4° C.

FluxOR™ Chloride-free Buffer (Component E)—5× Concentrate

-   -   Potassium sulfate (K₂SO₄) Concentrate (Component F)—125 mM in        water. Store at 4° C.    -   Thallium sulfate (Tl₂SO₄) Concentrate (Component G)—50 mM in        water. Store at 4° C.    -   DMSO (dimethyl sulfoxide, Component H)—1 ml (100%)

Reagent Preparation— FluxOR Working Solutions

-   -   1000× FluxOR™ Reagent: Reconstitute a vial of component A in 100        μl DMSO; Mix well; Store 10 μl aliquots at −20° C.    -   1× FluxOR™ Assay Buffer: Dilute Component B 10-fold with water;        Adjust pH to 7.4 with Hepes/NaOH; Filter and store at 4° C.    -   Probenecid/Assay Buffer: 100 ml of 1× FluxOR™ Assay Buffer; 1 ml        of reconstituted component D; Store at 4° C.    -   Loading Buffer (per microplate): 10 μl 1000× FluxOR™ Reagent;        100 μl component C; 10 ml Probenecid/Assay Buffer    -   Compound Buffer (per microplate): 20 ml Probenecid/Assay Buffer;        0.3 mM ouabain (10 mM ouabain in water can be stored in amber        bottle/aluminum foil at room temperature); Test compound    -   1× FluxOR™ Chloride-Free Buffer: Prepare 1× working solution in        water. Can be stored at room temperature    -   Stimulant Buffer (prepared at 5× final concentration in 1×        FluxOR™ Chloride-Free Buffer): 7.5 mM Thallium sulfate and 0.75        mM Potassium sulfate (to give a final assay concentration of 3        mM Thallium/0.3 mM Potassium). Store at 4° C. when not in use.        If kept sterile, this solution is good for months.

Assay Protocol—

The ROMK channel functional thallium flux assay is performed in 384wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded inPoly-D-Lysine microplates and kept in a 37° C.-10% CO₂ incubatorovernight. On the day of the experiment, the growth media is replacedwith the FluxOR™ reagent loading buffer and incubated, protected fromlight, at ambient temperature (23-25° C.) for 90 min. The loading bufferis replaced with assay buffer ±test compound followed by 30 minincubation at ambient temperature, where the Thallium/Potassiumstimulant is added to the microplate.

Step Protocol

-   1. Seed HEK-hKir1.1 cells (50 μl at 20,000 cells/well) in 384-well    PDL coated Microplates-   2. Allow cells to adhere overnight in humidified 37° C./10% CO₂    incubator-   3. Completely remove cell growth media from microplate and replace    with 25 μl loading buffer-   4. Incubate Microplate at room temperature, protected form light,    for 90 min-   5. Remove loading buffer and replace with 25 μl 1× Assay Buffer    ±test compound.-   6. Incubate microplate at room temperature, protected form light,    for 30 min-   7. At FLIPR-Tetra 384: Add stimulant (Thallium/Potassium) solution    to microplate and monitor fluorescence. Excitation=400 nm,    Emission=460 & 580 nm. Collect data for 10 min.

Data Calculation—

The fluorescence intensity of wells containing 3 μM of a standardcontrol ROMK inhibitor of the present invention is used to define theROMK-sensitive component of thallium flux. Fluorescence in the presenceof test compounds is normalized to control values to provide %fluorescence change. IC₅₀ values represent the concentration of compoundthat inhibits 50% of the ROMK thallium flux signal.

Assay Standard—

Normally, a control compound is included to support that the assay isgiving consistent results compared to previous measurements, althoughthe control is not required to obtain the results for the testcompounds. The control can be a compound of Formula I of the presentinvention, preferably with an IC₅₀ potency of less than 1 μM in thisassay. Alternatively, the control could be another compound (outside thescope of Formula I) that has an IC₅₀ potency in this assay of less than1 μM.

Representative examples of data collected for compounds of the presentinvention using the Electrophysiology and Thallium Flux Assays are shownin Table 1 below.

TABLE 1 Thallium Flux EP Thallium EP Assay Assay Flux Assay EXAM- IC₅₀IC₅₀ EXAM- Assay IC₅₀ PLE (μM) (μM) PLE IC₅₀ (μM) (μM) 1 0.274 0.117 12B0.057 0.105 2 1.207 13A 0.08633 0.031 3 0.865 13B 0.728 0.094 4A 0.0620.1203 14 0.241 0.108 4B 0.036 0.1173 15 0.103 0.2 5 0.737 0.348 160.6022 0.85 6 0.3586 0.201 17 0.5427 0.27 7A 0.1525 0.047 18A 0.13770.079 7B 0.566 2.404 18B 0.294 0.125 8A 0.402 0.062 19A 0.2897 0.046 8B0.662 19B 0.2847 0.066 9 0.234 20 1.048 10A 0.091 0.011 21 0.3194 1 10B0.076 0.184 22 0.9163 11A 0.093 0.039 23 0.54 1.8 11B 0.091 0.053 240.7885 12A 0.04 0.082 25 1.282

While the invention has been described with reference to certainparticular embodiments thereof, numerous alternative embodiments will beapparent to those skilled in the art from the teachings describedherein. Recitation or depiction of a specific compound in the claims(i.e., a species) without a specific stereoconfiguration designation, orwith such a designation for less than all chiral centers, is intended toencompass the racemate, racemic mixtures, each individual enantiomer, adiastereoisomeric mixture and each individual diastereomer of thecompound where such forms are possible due to the presence of one ormore asymmetric centers. All patents, patent applications andpublications cited herein are incorporated by reference in theirentirety.

1. A compound having structural Formula I:Z¹—Y¹—(CH₂)_(n1)—R—(CH₂)_(n2)—Y²—Z²  I or a pharmaceutically acceptablesalt thereof wherein: n1 and n2 can be individually either 0 or 1; Rrepresents a fused bicyclic or spirocyclic aliphatic diamine having oneof the following structures:

wherein R—I represents a 9-11-membered saturated bicyclic heterocyclicring system sharing 1 carbon atom, and R—II represents a 8-10-memberedsaturated bicyclic heterocyclic ring system sharing 2 carbon atoms,wherein each ring of R—I and each ring of R—II has 1 Nitrogen atom; R⁵is —H, —F with the proviso that —F is not attached to a carbon that isalso attached to nitrogen, —CH₃, —CF₃, —CHF₂, —CH₂F, or —CH₂OH, or R⁵represents di-substitution on a single carbon with two of —F with theproviso that —F is not attached to a carbon that is also attached tonitrogen or two of —CH₃; Z¹ is:

Z² is:

one of W¹ and W² is N and the other is CH; R¹ and R² are eachindependently —H, —F, —Cl, —Br, cyclopropyl, —C₁₋₃alkyl optionallysubstituted with 1-3 of —F, or —OC₁₋₃alkyl optionally substituted with1-3 of —F; one of R^(3a) and R^(3b) is —CN, tetrazolyl, or—S(O)₂C₍₁₋₃₎alkyl and the other is —H, —F, —Cl, —Br, —S—CH₃, —NH—CH₃,—O-cyclopropyl, —C₁₋₃alkyl optionally substituted with 1-3 of —F, or—OC₁₋₃alkyl optionally substituted with 1-3 of —F; one of R^(4a) andR^(4b) is CN, tetrazolyl, or —S(O)₂C₍₁₋₃₎alkyl and the other is —H, —F,—Cl, —Br, —S—CH₃, —NH—CH₃, —O-cyclopropyl, —C₁₋₃alkyl optionallysubstituted with 1-3 of —F, or —OC₁₋₃alkyl optionally substituted with1-3 of —F; R^(a), R^(aa), R^(b) and R^(bb) are each independently —H,—F, —Cl, —C₁₋₃alkyl optionally substituted with 1 to 3 of —F, or—OC₁₋₃alkyl optionally substituted with 1 to 3 of —F; R^(c) and R^(d)are each independently —H, —F, —Cl, —C₁₋₃alkyl optionally substitutedwith 1 to 3 of —F, or —OC₁₋₃alkyl optionally substituted with 1 to 3 of—F; and one of Y¹ or Y² is —CH(OH)—; and the other is —CH(OH)—; —C(O)—;or —S(O)₂—; provided that where Y¹ or Y² is —C(O)— or —S(O)₂—, then theadjacent n1 or n2, respectively, is 0; and provided further that wheren1 or n2 is 0, the adjacent Y¹ or Y² is —C(O)— or —S(O)₂—.
 2. Thecompound of claim 1 wherein R is:

and R⁵ is as defined in claim 1, or a pharmaceutically acceptable saltthereof.
 3. The compound of claim 1 wherein R is:

and R⁵ is as defined in claim 1, or a pharmaceutically acceptable saltthereof.
 4. The compound of claim 1 wherein: Z¹ is

and/or Z² is

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 5. The compound of claim 1 wherein: Z¹ is

and/or Z² is

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 6. The compound of claim 1 wherein R is:

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 7. The compound of claim 1 wherein R is:

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 8. The compound of claim 1 wherein R is:

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 9. The compound of claim 1 wherein R is:

and the other variables are as defined claim 1, or a pharmaceuticallyacceptable salt thereof.
 10. The compound of claim 1 wherein R is:

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 11. The compound of claim 1 wherein R is:

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 12. The compound of claim 1 wherein R is:

and the other variables are as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 13. A compound selected from:5,5′-{2,7-Diazaspiro[4.5]decane-2,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one);5-(1R)-2-(7-(6-(1H-tetrazol-1-yl)nicotinoyl)-2,7-diazaspiro[4.5]decan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one;5,5′-{2,8-diazaspiro[4.5]decane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one);6-({2-[(2R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]octahydro-5H-pyrrolo[3,4-c]pyridine-5-yl}carbonyl)pyridine-3-carbonitrile;5-((1R)-2-(5-(6-(1H-Tetrazol-1-yl)nicotinoyl)-1H-pyrrolo[3,4-c]_yridine-2(3H,3aH,4H,5H,6H,7H,7aH)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one;5,5′-{1,7-Diazaspiro[4.5]decane-1,7-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one);5,5′-{2,8-Diazaspiro[5.5]undecane-2,8-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one);5-((1R)-2-(8-(6-(1H-Tetrazol-1-yl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one;5-((1R)-1-Hydroxy-2-(8-(6-(methylsulfonyl)nicotinoyl)-2,8-diazaspiro[5.5]undecan-2-yl)ethyl)-4-methylisobenzofuran-1(3H)-one;5,5′-{Hexahydropyrrolo[3,4-b]pyrrole-1,5-diylbis[(1R)-1-hydroxyethane-2,1-diyl]}bis(4-methyl-2-benzofuran-1(3H)-one;6-(1-Hydroxy-2-{5-[(2R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)ethyl]hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl}ethyl)pyridine-3-carbonitrile;4-(1-((R)-2-Hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-5(1H)-ylsulfonyl)benzonitrile;4-(5-((R)-2-Hydroxy-2-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-ylsulfonyl)benzonitrile;6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrile;6-(1-Hydroxy-2-(1-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-2-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)ethyl)nicotinonitrile;5-((1R)-2-(5-(5-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one;5-((1R)-2-(5-(6-(1H-tetrazol-1-yl)picolinoyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one;5-((1R)-2-(5-(4-(1H-Tetrazol-1-yl)phenylsulfonyl)hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl)-1-hydroxyethyl)-4-methylisobenzofuran-1(3H)-one;5,5′-(1R,1′R)-2,2′-(1,7-Diazaspiro[4.4]nonane-1,7-diyl)bis(1-hydroxyethane-2,1-diyl)bis(4-methylisobenzofuran-1(3H)-one);5-((1R)-1-Hydroxy-2-(1-(3-methyl-4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-one;5-((1R)-1-Hydroxy-2-(1-(4-(1H-tetrazol-1-yl)phenylsulfonyl)-1,7-diazaspiro[4.4]nonan-7-yl)ethyl)-4-methylisobenzofuran-1(3H)-one;6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrile;6-(1-Hydroxy-2-(3-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-6-yl)ethyl)nicotinonitrile;6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrile;6-(1-Hydroxy-2-(6-((R)-2-hydroxy-2-(4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)-3,6-diazabicyclo[3.2.0]heptan-3-yl)ethyl)-4-methoxynicotinonitrile;or a pharmaceutically acceptable salt thereof.
 14. A pharmaceuticalcomposition comprised of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.
 15. Amethod for inhibiting ROMK comprising administering a compound of claim1 or a pharmaceutically acceptable salt thereof in a ROMK-inhibitoryeffective amount to a patient in need thereof.
 16. A method for causingdiueresis, natriuresis or both, comprising administering a compound ofclaim 1 or a pharmaceutically acceptable salt thereof in atherapeutically effective amount to a patient in need thereof.
 17. Amethod for the treatment of hypertension, heart failure or bothcomprising administering a compound of claim 1 or a pharmaceuticallyacceptable salt thereof in a therapeutically effective amount to apatient in need thereof.
 18. (canceled)
 19. A method for the treatmentor prophylaxis of one or more disorders selected from hepatic cirrhosis,acute and chronic kidney insufficiency, nephrotic syndrome, pulmonaryarterial hypertension, cardiovascular disease, diabetes, endothelialdysfunction, diastolic dysfunction, stable and unstable angina pectoris,thromboses, restenosis, myocardial infarction, stroke, cardiacinsufficiency, pulmonary hypertonia, atherosclerosis, ascitis,pre-eclampsia, cerebral edema, nephropathy, hypercalcemia, Dent'sdisease, Meniere's disease or kidney stones comprising administering acompound of claim 1 or a pharmaceutically acceptable salt thereof in atherapeutically or prophylactically effective amount as appropriate, toa patient in need therof.